Methods, systems, and devices for detecting and identifying microorganisms in microbiological culture samples

ABSTRACT

Provided herein are methods, systems, and devices for detecting and/or identifying one or more specific microorganisms in a culture sample. Indicator particles, such as surface enhanced Raman spectroscopy (SERS)-active nanoparticles, each having associated therewith one or more specific binding members having an affinity for the one or more microorganisms of interest, can form a complex with specific microorganisms in the culture sample. Further, agitating magnetic capture particles also having associated therewith one or more specific binding members having an affinity for the one or more microorganisms of interest can be used to capture the microorganism-indicator particle complex and concentrate the complex in a localized area of an assay vessel for subsequent detection and identification. The complex can be dispersed, pelleted, and redispersed so that the culture sample can be retested a number of times during incubation so as to allow for real-time monitoring of the culture sample.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The presently disclosed subject matter relates to methods, systems, anddevices for detecting, identifying, and quantifying microorganisms in aculture sample. More particularly, the subject matter relates to the useof indicator particles to detect and identify one or more microorganismsin a biocontained sample capable of supporting growth of microorganisms.

2. Background of the Invention

The ability to detect low levels of microorganisms, including pathogens,in a microbiological culture in clinical samples (e.g., blood, stool,urine, etc.) has gained significant importance in recent years.Similarly, microbiologial culture is important to public health todetect microorganisms, including pathogens, in industrial samples suchas food, cosmetics, and pharmaceuticals. The ability to detect suchmicroorganisms not only provides techniques for treating those who havealready been exposed, but also to instances where exposure can beprevented, such as when testing food samples.

Foodborne illnesses significantly impact society, not only with respectto health, but also health-care costs. The CDC has estimated that eachyear about 1 in 6 Americans (or 48 million people) gets sick, 128,000are hospitalized, and 3,000 die of foodborne diseases (seehttp://www.cdc.gov/foodsafety/facts.html). It has also been estimatedthat foodborne illnesses contribute to $152 billion in health-relatedexpenses each year in the U.S., particularly for bacterial infectionscaused by Campylobacter spp., Salmonella, Listeria monocytogenes and E.coli (seehttp://www.producesafetyproject.org/admin/assets/files/Health-Related-Foodborne-Illness-Costs-Report.pdf-1.pdf).

The current level of food safety found in the U.S. is the result ofGovernment regulations combined with industry self-monitoring influencedby market incentives, such as legal liability, brand value, reputation,and the desire to sell more food product. In the U.S., the primaryagencies responsible for food safety are the U.S. Department ofAgriculture (USDA) Food Safety and Inspection Services (FSIS), which isresponsible for the safety of meat, poultry, and processed egg products,and the Food and Drug Administration (FDA), which is responsible forvirtually all other foods. In 1996, USDA's FSIS promulgated the pathogenreduction hazard analysis critical control point (PR/HACCP) rule, which,for example, mandates generic E. coli testing by slaughter plants. OtherFSIS regulations enforce zero limits for two deadly pathogens—Listeriamonocytogenes in ready-to-eat meat and poultry and E. coli O157:H7 inground beef (seehttp://www.ers.usda.gov/briefing/foodsafety/private.htm). Recently, theFood Safety Modernization Act was approved by Congress, the urgency forthis legislation being underscored by continued outbreaks of foodborneillness over the last several years—from spinach to peppers to peanuts.

Food testing may occur on food samples themselves, either end productmaterials, intermediates, or incoming raw materials. In addition, HACCP(Hazard Analysis and Critical Control Point) plans are implemented tocontrol the production environment so as to minimize the risk ofintroduction of pathogens into the food sample. As part of many HACCPplans, environmental samples are acquired from surfaces, floors, drains,and processing equipment and then analyzed for the presence and absenceof pathogenic organisms. If a pathogen is detected, it may be isolatedand subjected to further confirmatory testing.

Today, all food pathogen testing conducted entails a culture step toenrich the potentially low levels of microorganisms contained in asample. Following culture of the sample, a portion is removed and testedfor the presence of pathogens. Pathogen testing after culture can bedone by immunoassays (e.g., bioMerieux's Vidas® automated ELISA platformor SDIX's RapidChek® lateral flow assays) or by PCR-based tests (e.g.,DuPont Qualicon's BAX® system, Bio-Rad's iQ-Check™ system). If apathogen is present in the starting sample, the culture step canincrease the concentration of the pathogen as high as 1.0E8-1.0E9cfu/mL, so that opening the sample after culture exposes both the userand the environment to a risk of contamination. This exposure inhibitsmany food producers from conducting pathogen testing on-site, insteadchoosing to send samples to external laboratories for testing. Inaddition, since it is unknown which samples contain pathogens and atwhat levels, food safety test protocols use lengthy culture times toensure that the worst case scenario of one damaged pathogen is givensufficient time to grow to a detectable concentration. As a consequence,samples with higher pathogen loadings are cultured longer than may bestrictly necessary, leading to a delay in time to results. There is thusa need in the field for pathogen test methods that minimize time toresults and reduce the risk of exposure of the facility and personnel tocultured pathogens.

Similar concerns are present for clinical samples such as blood. Sincethe mid-1980s, along with the expanding size of the immunocompromisedpatient population, the incidence of septicemia caused by opportunisticpathogens, such as yeast, fungi, and mycobacteria, has risen.Bacteremia, the presence of bacteria in the blood stream, and fungemia,the presence of fungi or yeasts in the blood stream, typically aredetected by collecting a venous blood sample and disposing the bloodsample in a blood culture bottle containing a growth medium suitable forpromoting growth of the bacteria or fungi of interest. See generally,Reimer et al., “Update on Detection of Bacteremia and Fungemia,”Clinical Microbiology Reviews 10(3), 444-465 (1997). The blood culturesample can then be incubated for a period of time and checkedintermittently for an indication of bacterial or fungal growth.

Instrumented methods known in the art for monitoring bacterial or fungalgrowth in blood culture bottles typically detect changes in the carbondioxide and/or oxygen concentration in the blood culture bottle. Theseinstruments detect the presence and absence of microorganisms but arenot specific as to the particular type of organism present. For anominally sterile sample such as blood, detection of a microorganism inthe sample can be indicative of severe disease. However, the positiveresult is considered to be a partial or preliminary result and istypically not actionable. As optimum treatment of the disease relies onidentification of the organism and determination of its antibioticsusceptibility, laboratory personnel must be available to advancepositive cultures to full identification (ID) and antimicrobialsusceptibility testing (AST). Identification of the organism requiresaccessing of the positive blood culture sample by laboratory personnelfor further sample work-up.

Sample work-up following a positive blood culture result, i.e., a resultindicating the presence but not identity of a microorganism, oftenincludes categorization of the microorganism into one of two broadclasses of organisms: Gram positive or Gram negative. Blood cultureassays based on the detection of CO₂ or O₂ during the culture processcannot distinguish between pathogenic organisms, such as S. aureus, andcontaminants, such as S. epidermidis since these methods are sensitiveonly to growth and absence of growth. Classification and identificationof organisms is performed following the detection of growth in a bloodculture sample. For example, kits are available for differentiatingbetween Staphylococcus and Streptococcus species and other organisms.Kits also are available for differentiating between organisms, such asS. aureus and S. epidermidis. These kits, however, require removing atleast an aliquot of a blood culture sample from the blood culture bottleand other procedures that can potentially expose the operator to thepathogen or destroy a portion of the blood culture that could be usedfor other analyses. They also typically require that trained laboratorystaff are available to conduct the tests, potentially leading to a delayin actionable clinical results in the event that a blood culture samplegoes positive when laboratory personnel are unavailable to conductadditional testing (e.g., in hospitals that operate only a singleshift.)

While instruments exist today to detect the presence or absence ofmicroorganisms in blood (e.g., by use of a carbon dioxide or oxygensensor), these instruments are not typically useful in non-sterilesamples such as stool or food samples. For samples such as, for examplefood, there is expected to be a significant concentration of benignmicroorganisms, and so detection of organisms by carbon dioxide oroxygen sensors is not inherently useful. For a food sample, it iscritical to detect the presence of low levels of pathogenic organisms ina background of high benign microflora to avoid the spread of foodborneillnesses.

Therefore, there is a need for methods, systems, and devices fordetecting not only the presence or absence of organisms during theculture step of nominally sterile samples, but also identification ofthe organisms. For non-sterile samples, such as stool and food, there isalso a need for methods, systems, and devices for identifyingpotentially harmful organisms in a culture in a biocontained manner.Such methods, systems and devices minimize user intervention, therebyminimizing time, trained personnel, plus potential exposure of personneland environment to the pathogen.

SUMMARY OF THE INVENTION

Embodiments of the presently disclosed subject matter provide methods,systems, and devices for detecting the presence, amount, and/or identityof specific microorganisms in a microbiological culture. According toone embodiment, the presently disclosed assays can be performed withinthe culture vessel, so that detection and/or identification of specificmicroorganisms occur in conjunction with culture, without the need foruser intervention. One or more microorganisms can be identified within asingle culture. The culture vessel can be fully biocontained so that thegrowth of the microorganism and microorganism detection andidentification can occur without exposing either the user or thesurrounding environment. Moreover, due to the biocontainment of theculture, the analysis of the culture may occur without the need for theuser to access the culture or wash the culture.

Optically active indicator particles, such as Surface Enhanced RamanScattering (SERS)-active nanoparticles, each having associated therewithone or more specific binding members having an affinity for the one ormore microorganisms of interest, can form a complex with specificmicroorganisms in the microbiological culture sample. Thus, theoptically active indicator particles can be any particle capable ofproducing an optical signal that can be detected in a culture samplewithout wash steps. Further, magnetic capture particles, also havingassociated therewith one or more specific binding members having anaffinity for the one or more microorganisms of interest, which can bethe same or different from the specific binding members associated withthe indicator particles, can be used to capture themicroorganism-indicator particle complex and concentrate the complex ina localized area of an assay vessel for subsequent detection.Importantly, embodiments of the presently disclosed methods, systems,and devices allow “real-time” detection and identification ofmicroorganisms in a sample in which active growth of the microorganismis occurring. Samples may include microbiological cultures comprising agrowth medium and a clinical sample from a human or animal (domestic orstock) such as blood, stool, urine, or cerebral spinal fluid. Samplesmay also include microbiological cultures comprising a growth medium andan industrial sample such as food, dairy, beverage, water,environmental, agricultural products, personal care products (includingcosmetics), biotechnology, or pharmaceuticals. Importantly, the assaycan be conducted in a biocontained manner without exposure of the useror environment to the sample (“closed system”) and can provideautomated, around the clock, detection and identification ofmicroorganisms by monitoring the assay signal over time as the cultureprogresses. The combination of detection and identification withmicrobiological culture can lead to earlier availability of actionableresults.

Detection of microorganisms by the present invention can be performedeither directly or indirectly. For direct detection of micorganismsgrowing in culture, the specific binding members associated with themagnetic capture particles and indicator particles can have an affinityfor the largely intact microorganism, e.g. by binding to the surface ofbacteria or yeast. For indirect detection, the binding membersassociated with the magnetic capture particles and indicator particlesmay have an affinity for byproducts of the microorganism. Examples ofbyproducts could include but are not limited to secreted proteins,toxins, and cell wall components. Direct and indirect detection modesmade be used alone or in combination.

According to another embodiment of the present invention, a vessel formetering a desired amount of culture sample is provided. The vesselincludes a container for receiving a culture sample therein, wherein thecontainer has an open end and a closed end. The vessel also includes alid configured to engage the open end of the container in a fluid-tightconnection. In addition, the vessel includes a basket coupled to the lidand including one or more reservoirs, wherein the basket is disposedbetween the open end and the closed end of the container. Where aplurality of reservoirs is used, each reservoir is configured to hold adifferent volume of culture sample. Moreover, the vessel includes one ormore needle assemblies engaged with the lid, wherein the needle assemblyincludes a needle extending within a respective reservoir. Each needleis configured to selectively withdraw a sample contained in a respectivereservoir, wherein each needle is further configured to engage a vialfor a biocontained transfer of the sample from the reservoir to thevial. Thus, the vessel may be suitable for metering a desired amount ofsample for two different assays (e.g., Salmonella or Listeria) in asingle container, while facilitating transfer of the sample to adetection vial in a biocontained manner. In another embodiment of thepresent invention, the assay vial for receiving a sample is enclosed bya stopper or septum and cap configured to retain a vacuum. Uponconnection of the assay vial cap with a compatible port containing aneedle on the metering vessel, the sample is transferred in abiocontained fashion. The vial cap contains features to retainexternally expressed fluid from the transfer and protect the user fromcontact with transfer surfaces.

Another embodiment of the present invention is directed to a system forautomatically processing a plurality of tubes containing a culturesample. The system includes an incubator for receiving a plurality ofsample tubes therein, wherein the incubator is configured to incubatethe sample tubes at a predetermined temperature. For example, the tubesmay be positioned horizontally and adjacent to each other. The incubatormay be configured to incubate different assays at different temperaturesaccording to one embodiment. The system further includes a firsttranslational device (e.g., a “Y-stage” for movement along a Y-axis)coupled to the tray and configured to move the sample tubes within theincubator, wherein the first translational device is further configuredto move the sample tubes from the incubator to a detection zone and toagitate the sample tubes within the detection zone. For instance, thefirst translational device may move the samples tubes along theirlongitudinal axes. The system also includes a magnet assembly configuredto apply a magnetic field to the plurality of sample tubes within thedetection zone, as well as an optical device configured to interrogateeach of the plurality of sample tubes within the detection zone fordetecting one or more microorganisms. The system includes a secondtranslational device (e.g., an “X-stage” for movement along an X-axis)coupled to the optical device and configured to move the optical devicewithin the detection zone for interrogating each of the sample tubes.The system may also include a third translational device (e.g., a“Z-stage” for movement along the Z-axis) coupled to the magnet assemblyand the optical device and configured to move the magnet assembly andoptical device within the detection zone to access another tray of tubesstacked vertically above the first tray. Thus, the system provides anautomated and high-throughput system for processing a plurality ofsamples in real time during incubation of the culture tubes.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying drawings, whichare not necessarily drawn to scale, and wherein:

FIG. 1 is a schematic diagram showing a method of detecting andidentifying a microorganism in a culture sample according to anembodiment of the invention.

FIG. 2 is a schematic diagram showing an enrichment vessel and adetection vial for containing and transferring a culture sampleaccording to one embodiment of the present invention.

FIG. 3 is a schematic diagram showing a method of intermittent detectingand identifying of a microorganism in a culture sample according to anembodiment of the invention.

FIG. 4 is a schematic diagram showing a method of real-time detectingand identifying of a microorganism in a culture sample according to anembodiment of the invention.

FIGS. 5A-5E illustrate various views of an enrichment vessel accordingto one embodiment of the present invention.

FIG. 6 is a cross-sectional view of an enrichment vessel according toone embodiment of the present invention.

FIG. 7 is an exploded view of an enrichment vessel according to oneembodiment of the present invention.

FIG. 8 is a bottom view of lid for an enrichment vessel according to oneembodiment of the present invention.

FIGS. 9A-9C are various views of a basket for an enrichment vesselaccording to one embodiment of the present invention.

FIGS. 10A and 10B illustrate a cap for a detection vial according to oneembodiment of the present invention.

FIGS. 11A and 11B illustrate a cap for a detection vial according to oneembodiment of the present invention.

FIG. 12 is a cross-sectional view of a cap engaging a detection vialaccording to one embodiment of the present invention.

FIGS. 13A and 13B are a perspective view and an exploded view of a capengaging a detection vial according to an embodiment of the presentinvention.

FIGS. 14A and 14B are a perspective view and an exploded view of a capengaging a detection vial according an embodiment of the presentinvention.

FIG. 15 is a cross-sectional view of detection vials engaging anenrichment vessel according to an embodiment of the present invention.

FIGS. 16 and 17 are cross-sectional views of a detection vial engagingan enrichment vessel according to an embodiment of the presentinvention.

FIG. 18 is a schematic diagram showing a magnetic captureparticle-microorganism-SERS-active indicator particle complex within aculture bottle according to an embodiment the invention.

FIG. 19 depicts a SERS-active indicator particle according to oneembodiment of the present invention.

FIG. 20 depicts a SERS-active indicator particle according to oneembodiment of the present invention.

FIG. 21 depicts a SERS-active indicator particle according to oneembodiment of the present invention.

FIG. 22 shows a representative SERS spectrum of a SERS-active indicatorparticle having associated therewith a 4,4′-dipyridyl (DIPY)Raman-active dye according to an embodiment of the invention.

FIG. 23 shows a representative SERS signal plotted over culture time forSalmonella according to an embodiment of the invention.

FIG. 24 depicts a system for real-time monitoring of microorganismgrowth according to an embodiment of the invention.

FIG. 25 depicts a system for real-time monitoring of microorganismgrowth according to another embodiment of the invention.

FIGS. 26-29 illustrate various views of a system for real-timemonitoring of microorganism growth according to an additional embodimentof the invention.

FIG. 30 is a perspective view of a tray for holding sample tubesaccording to an embodiment of the present invention.

FIG. 31 illustrates sequential steps for loading sample tubes into atray, loading the tray into an incubator, and removing the trays fromthe incubator, according to an embodiment of the present invention.

FIG. 32 is a perspective view of a tray for holding sample tubesaccording to another embodiment of the present invention.

FIGS. 33A-33C are partial views of trays for holding sample tubesaccording to various embodiments of the present invention.

FIG. 34 is a perspective view of an incubator according to an embodimentof the present invention.

FIGS. 35-39 are various cross-sectional views of the system shown inFIGS. 26-29.

FIG. 40 is an enlarged view of a rear door of an incubator according toan embodiment of the present invention.

FIG. 41 is a perspective view of an X-stage according to an embodimentof the present invention.

FIG. 42 is a perspective view of a magnet assembly, an X-stage, and aZ-stage according to an embodiment of the present invention.

FIG. 43 is a side view of a magnet assembly, a pelleting/read assembly,an X-stage, a Y-stage, and a Z-stage in a lowered position according toan embodiment of the present invention.

FIG. 44 is another perspective view of the system shown in FIGS. 26-29.

FIGS. 45 and 46 are partial perspective views of a magnet assembly, apelleting/read assembly, and an X-stage according to one embodiment ofthe present invention.

FIG. 47 is partial perspective view of a magnet assembly and a Y-stageaccording to one embodiment of the present invention.

FIGS. 48A-48B are perspective views of a system for real-time monitoringof microorganism growth enclosed in a cabinet according to embodimentsof the present invention.

FIG. 49 illustrates a method for agitating and pelleting a culturesample according to an embodiment of the present invention.

FIG. 50 depicts a pelleting and optical system according to anembodiment of the invention.

FIGS. 51 and 52 illustrate alternative magnet arrangements for pelletinga culture sample according to embodiments of the present invention.

FIG. 53 depicts a multiplexed detection of S. aureus and S. epidermidisaccording to an embodiment of the invention.

FIG. 54 shows the results of an experiment in which time to detection ofE. coli growth was compared for blood culture samples with and withoutthe SERS HNW reagents suitable for use in the various embodiments of theinvention.

FIG. 55 shows a graph in which the growth of Salmonella entericasubspecies enterica serovar Typhimurium, henceforth referred to asSalmonella Typhimurium (or other Salmonella serovar name), was monitoredin relation to the effect of pelleting thereon according to anembodiment of the invention.

FIG. 56 shows a graph illustrating the effect of pelleting onmicroorganism growth according to an embodiment of the invention.

FIG. 57 illustrates an image of a SERS-magnetic bead precomplex (PC) inwater after pelleting with a fixed magnet according to one embodiment.

FIGS. 58A-58B are images of PC pellet formation in SDIX Salmonellasecondary media using a fixed magnet and different agitation frequenciesaccording to one embodiment.

FIGS. 59A-59B are images of PC pellet formation in SDIX Salmonellasecondary media using a coupled magnet and different agitationfrequencies according to one embodiment.

FIG. 60 shows a graph in which time to detection of C. albicans in bloodwas compared using a singleplex SERS detection according to anembodiment of the invention.

FIG. 61 shows a graph in which time to detection of C. albicans in bloodwas compared using a multiplex SERS method according to an embodiment ofthe invention.

FIG. 62 shows a graph in which time to detection of E. coli and S.epidermidis in blood was compared using a multiplex SERS methodaccording to an embodiment of the invention.

FIG. 63 illustrates a graph of real-time detection of E. coli in bloodwith aerobic media and antibiotic absorbing resins according to anembodiment of the present invention.

FIG. 64 shows a graph of the detection of E. coli in blood for differentsample volumes according to an embodiment of the present invention.

FIG. 65A shows a SERS curve with images captured at various times duringsecondary enrichment of Salmonella Typhimurium according to oneembodiment.

FIG. 65B shows a SERS curve with images captured at various times duringsecondary enrichment for a negative sample according to one embodiment.

FIG. 65C shows a SERS curve with images captured at various times duringsecondary enrichment of Salmonella Typhimurium according to oneembodiment.

FIG. 66 shows overlaying SERS curves for different agitation ratesduring secondary enrichment of Salmonella Typhimurium according to oneembodiment.

FIG. 67 shows images of pellets for a positive sample and a negativesample, respectively, according to an embodiment of the presentinvention.

FIGS. 68A-68C illustrate SERS curves for the real-time detection of E.coli during culture in food samples according to embodiments of thepresent invention.

FIG. 69 illustrates SERS curves for the real-time detection ofSalmonella Enteritidis during culture in food samples according to anembodiment of the present invention.

FIG. 70 illustrates SERS curves for the real-time detection of Listeriaswabbed from stainless steel during culture according to an embodimentof the present invention.

FIG. 71 shows a flowchart of phases for the detection of SalmonellaTyphimurium using linear agitation according to an embodiment of thepresent invention.

FIG. 72 illustrates overlaying SERS curves during secondary enrichmentfor Salmonella Typhimurium, Salmonella Enteritidis, and negative samplesaccording to an embodiment of the present invention.

FIG. 73 shows images of pellets formed during secondary enrichment ofSalmonella Typhimurium according to an embodiment of the presentinvention.

FIG. 74 shows SERS curves obtained from rocking agitation and linearagitation during secondary enrichment of S. Enteritidis and S. Kentuckyaccording to one embodiment.

FIG. 75 shows images of sample tubes containing S. aureus and S.epidermidis in EDTA rabbit plasma, with and without SERS reagents,according to one embodiment of the present invention.

FIG. 76 shows images of latex agglutination assays with S. aureus and S.epidermidis, with and without SERS reagents, according to one embodimentof the present invention.

FIG. 77 is a magnified image of gram staining of a mixture of magneticparticles and SERS tags according to one embodiment of the presentinvention.

FIG. 78 is a magnified image of gram stained controls of S. aureus andE. coli with magnetic particles and SERS tags according to oneembodiment of the present invention.

FIG. 79 shows images of CHROMagar S. aureus plates streaked with a bloodculture of S. aureus and S. epidermdis with SERS reagents according toembodiments of the present invention.

FIG. 80 is an image of an agar plate streaked with a blood culture of E.coli with Sensi-disc™ test discs according to an embodiment of thepresent invention.

FIG. 81 is a table showing zone diameter measurements for E. coli, withand without reagents, according to an embodiment of the presentinvention.

FIG. 82 is a table showing a summary of the results of manual antibioticsusceptibility testing using BD Sensi-discs™ and various microorganismswith and without SERS reagents, according to one embodiment of thepresent invention.

FIG. 83 shows images of agar plates streaked with a blood culture of E.coli and C. albicans, with and without reagents, overlaid withanti-fungal BD Taxo™ discs, according to an embodiment of the presentinvention.

FIG. 84 is a table showing images of pellets formed in Salmonellasecondary media using different agitation frequencies and pelletingtimes, according to an embodiment of the present invention.

FIG. 85 is a table showing the effect of agitation frequency on pelletdispersion, according to an embodiment of the present invention.

FIG. 86 illustrates an enrichment vessel according to another embodimentof the present invention.

FIG. 87 is a cross-sectional view of a syringe according to oneembodiment of the present invention.

FIG. 88 is a cross-sectional view of a syringe engaged with anenrichment vessel according to one embodiment of the present invention.

FIGS. 89A-89C are enlarged cross-sectional views of a syringe engagedwith an enrichment vessel according to various embodiments of thepresent invention.

FIGS. 90A and 90B are enlarged cross-sectional views of a syringeaccording to one embodiment of the present invention.

FIG. 91 is a cross-sectional view of a syringe and a perspective view ofa plunger according to one embodiment of the present invention.

FIG. 92 is a cross-sectional view of a syringe and a perspective view ofa plunger according to another embodiment of the present invention.

FIGS. 93-95 illustrate reconstitution stations according to variousembodiments of the present invention.

FIG. 96 is an image of fabricated fluorescent silica nanoparticlesaccording to one embodiment of the present invention.

FIG. 97 shows a graph depicting the signal intensity of fabricatedfluorescent silica nanoparticles and conventional SERS tags according toone embodiment of the present invention.

FIG. 98 shows a graph depicting the signal intensity over time offabricated fluorescent silica nanoparticles and conventional SERS tagsfor detecting the presence of Listeria in spinach according to oneembodiment of the present invention.

FIG. 99 shows a graph depicting the signal intensity over time offabricated fluorescent silica nanoparticles and conventional SERS tagsfor detecting the presence Listeria in cabbage according to oneembodiment of the present invention.

FIG. 100 is a perspective view of a container for an enrichment vesselaccording to one embodiment of the present invention.

FIG. 101 is a top view of lid for an enrichment vessel according to oneembodiment of the present invention.

FIG. 102 is a bottom view of the lid shown in FIG. 101.

FIG. 103 is a bottom perspective view of the lid shown in FIG. 101.

FIG. 104 is a side view of the lid shown in FIG. 101.

FIG. 105 is a cross-sectional view of the lid shown in FIG. 101.

FIG. 106 is a side view of a basket for an enrichment vessel accordingto one embodiment of the present invention.

FIG. 107 is a top view of the basket shown in FIG. 106.

FIG. 108 is a bottom view of the basket shown in FIG. 106.

FIG. 109 is a perspective view of the basket shown in FIG. 106.

DETAILED DESCRIPTION OF THE INVENTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Drawings, in which some,but not all embodiments of the presently disclosed subject matter areshown. Many modifications and other embodiments of the presentlydisclosed subject matter set forth herein will come to mind to oneskilled in the art to which the presently disclosed subject matterpertains having the benefit of the teachings presented in the foregoingdescriptions and the associated Drawings. Therefore, it is to beunderstood that the presently disclosed subject matter is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims. Although specific terms are employed herein, they areused in a generic and descriptive sense only and not for purposes oflimitation.

The terms “a,” “an,” and “the” refer to “one or more” when used in thisapplication, including the claims. Thus, for example, reference to “asample” includes a plurality of samples, unless the context clearly isto the contrary (e.g., a plurality of samples), and so forth.

Throughout this specification and the claims, the words “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise.

As used herein, the term “about,” when referring to a value is meant toencompass a specified value and variations thereof. Such variations maybe, in some embodiments ±100%, in some embodiments ±50%, in someembodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, insome embodiments ±1%, in some embodiments ±0.5%, and in some embodiments±0.1% from the specified amount, as such variations are appropriate toperform the disclosed methods or employ the disclosed compositions.

Further, when an amount, concentration, or other value or parameter isgiven as either a range, preferred range, or a list of upper preferablevalues and lower preferable values, this is to be understood asspecifically disclosing all ranges formed from any pair of any upperrange limit or preferred value and any lower range limit or preferredvalue, regardless of whether ranges are separately disclosed. Where arange of numerical values is recited herein, unless otherwise stated,the range is intended to include the endpoints thereof, and all integersand fractions within the range. It is not intended that the scope of thepresently disclosed subject matter be limited to the specific valuesrecited when defining a range.

The embodiments of the present invention provide systems and methodswhich utilize indicator particles (e.g., surface enhanced Ramanscattering (SERS)-active indicator particles), for detecting and/oridentifying one or more microorganisms in a bacterial culture sample bya Homogeneous No Wash assay (HNW). More specifically, embodiments of theinvention describe techniques for monitoring the concentration ofmicroorganism in “real-time” as the microorganism level increases overtime within a sample. The indicator particles have associated therewithone or more specific binding members having an affinity for the one ormore microorganisms under test. When contacted with a microbiologicalculture sample containing one or more microorganisms of interest, acomplex, generally referred to herein as an indicatorparticle-microorganism complex, between the one or more microorganismsof interest and the indicator particle with associated specific bindingmember can be formed. The indicator particle-microorganism complex canbe captured by a magnetic capture particle and concentrated to form apellet in a localized area (i.e., a “measurement zone”) for detection bymeasuring the signal (e.g., SERS spectrum) and/or a visual inspection ofan image of the pellet. The term “pellet”, as used herein, is not meantto be limiting and in one embodiment, refers to a collection of aplurality of indicator particles and magnetic capture particles locatedin a localized area facilitated by application of a magnetic field,wherein the pellet is detectable using visual, optical, or othersuitable means. The pellet may also include microorganisms capturedtherebetween, if present, and other components and/or microorganisms maybe non-specifically attached to the magnetic particles. The pellet maybe temporarily formed in that the pellet may be dispersed upon removalof the magnetic field as discussed in greater detail below.

Furthermore, the various embodiments of the invention pertain to theability to conduct the HNW assay repeatedly within the samemicrobiological culture sample, by forming, dispersing, and reformingthe pellet over time. This enables the concentration of a particularanalyte to be monitored real-time within a microbiological culturesample and is particularly valuable when the microorganism concentrationis changing over time, e.g. in response to bacterial growth. Moreparticularly, embodiments of the invention pertain to the ability toconduct the HNW assay within a microbiological culture vessel, therebysimultaneously detecting and identifying a microorganism as it grows. Inaddition, the technique can be used in conjunction with other methods ofmonitoring the culture sample (such as gas sensor or image analysis).

According to an embodiment of the invention, a microbiological cultureof the sample is conducted in a vessel that also contains the HNWreagents. The culture vessel is inserted into an instrument that allowsincubation at a controlled temperature and contains optical devices(e.g., Raman optics, a Raman laser, and a spectrometer). At regular timeintervals during the culture, a magnetic field is applied, and the SERSsignal is read from the magnetic pellet. The pellet is dispersed betweenreadings to allow continued interactions of the reagents with thesample. As the target organism concentration increases throughout theenrichment process, detection and identification of the microorganism bythe SERS technology occurs as soon as the microorganism concentrationreaches the detection threshold of the technology. The ability tocontinuously monitor the SERS signal during culture ensures that theminimal required culture time is used and that the instrument canautomatically alert the user when a microorganism is detected andidentified.

A further embodiment uses a camera to monitor the formation and size ofa pellet during a HNW assay which contains conjugated indicatorparticles and magnetic beads and the targeted pathogen within a culturevessel. Images show that pellet size increases, and in some cases thepellet disappears, from the camera view as the HNW assay progresses. Thegrowth in pellet size and/or disappearance of the pellet is anindication of the presence of the targeted pathogen. Images capturedduring analysis of samples that contain conjugated indicator particlesand magnetic beads with no pathogen show no change in pellet size and nopellet disappearance. This method of detection can be used alone or inconjunction with another detection method.

I. General Considerations for Detection and Identification ofMicroorganisms in a Microbiological Culture Sample

As used herein, the term “microbiological culture sample” refers to acomposition comprising a “clinical” or an “industrial” sample with thepotential of containing microorganisms that is disposed in, admixed, orotherwise combined with a culture medium, e.g., a blood culture broth,capable of supporting the growth of one or more microorganisms suspectedof being present in the sample. More particularly, embodiments of thepresently disclosed subject matter provide methods, systems, and devicesfor detecting microorganisms in a microbiological culture samplecomprising a media capable of supporting microorganism growth in eithera clinical sample, such as blood, stool, urine or cerebral spinal fluid,or in an industrial product sample, such as food, environmental swabs orsponges, water, cosmetics, hygiene products, pharmaceuticals, or otherproducts intended for use or consumption by animals or humans.

Detecting and/or identifying microorganisms in microbiological culturesamples, especially with optical or spectrometric methods, can presentmany challenges due to the complexity of the sample matrix. Clinicalsamples, particularly those such as blood or stool, are opticallyabsorptive, making it difficult to detect optical or spectral signalswithout wash or lysis steps to remove optically interfering componentsof the original samples. Industrial samples, such as, for example foodor cosmetic samples, may be optically absorptive, again requiring washor lysis steps to remove optical interferents in the original sample.Although the application of SERS to detecting mammalian cells andmicroorganisms and the diagnostic application of SERS-active indicatorparticles to detecting a variety of analytes in the presence of bloodand food samples has been reported, the application of SERS-activeindicator particles to monitor bacteria and fungi concentrations in“real time” as the concentrations change due to microorganism growth hasnot been reported. As used herein, “real time” is not meant to belimiting and may refer to monitoring the culture sample continuously orin predetermined increments of time. For example, the culture sample maybe tested repeatedly in predetermined increments of time (e.g., every 30minutes, 1 hour, etc.) over a predetermined incubation period withoutopening the sample tube thereby maintaining biocontainment of thesample. “Biocontainment”, as used herein, is also not meant to belimiting and may refer to the culture sample being in a closed systemsuch that the surrounding environment outside of the container in whichthe culture sample is confined is not exposed to the microorganismsbeing cultured.

Further, the presently disclosed methods allow for the diagnostic use ofindicator particles in microbiological cultures in a manner that doesnot inhibit the growth of the microorganism under detection.

Current methods of detecting the presence or absence of pathogens duringmicrobiological growth, e.g. blood culture cabinets, do not specificallydetect organisms, but rather a non-specific product of metabolism (e.g.,carbon dioxide). Therefore, these sensors can potentially be falselytriggered by carbon dioxide produced by other processes, such asoxidation, degradation, and respiration of the blood culture cells(e.g., mammalian cells) that are normal flora in a blood sample. Thissignificant ‘blood background’ signal is an important noise source thatcomplicates positivity algorithms and decreases overall analyticalsensitivity. The signal generated from a specific binding event, asdescribed in the presently disclosed methods, will be a clear indicatorthat a pathogen is present and will not likely be misinterpreted.

The various embodiments of this invention allow continuous growth,detection and identification all within the geometry of a single vial.The SERS HNW technology enables a culture system capable of providinground the clock (24 hours/7 days a week) alerts on growth positivesamples along with additional identifying information (e.g., gram staininformation or identification). In contrast to blood culture systemscurrently on the market which detect the absence or presence of growth,the SERS HNW assay can provide identification of the microorganism orclass of microorganisms. Antibodies conjugated to the SERS and magneticparticles can be selected to specifically identify gram positive versusgram negative bacteria. Importantly, the inherent multiplexingcapabilities of the SERS technology are key for the blood culture andindustrial applications.

Existing gas based sensors such as those used in blood culture cabinetsare unsuitable for detecting the presence of pathogenic microorganismsin samples (e.g. stool, food, or environmental samples) wherein there isan expected high level of background benign microorganisms. There arecurrently no known methods for real-time pathogen detection within afood or an environmental sample, because these types of samplestypically have background (benign) microorganisms that also grow duringculture, so a growth based sensor cannot distinguish between growth ofthe background organisms and growth of the target pathogen.

In addition, existing methods for microorganism identification require acombination of sample preparation and/or wash steps to removeinterfering components, minimize background signal, and/or generate asample that is optically transparent. Because of the sample preparationand wash requirements, these methods cannot be applied within an ongoingculture.

The SERS-HNW assay overcomes the problems of the need for wash steps bygenerating a Raman signal that can be read in a dirty or non-isolatedsample. It also enables multiplexed detection and identification incomplex matrices, thereby making it suitable for the multiplexeddetection of blood stream infections or food pathogens. These attributesof the HNW assay have been previously disclosed. However, in all knownprevious disclosures, the HNW assay was applied a single time to asingle sample, i.e., one pellet was formed and read to generate the“answer” (identification+detection). There has been no indication thatthe conduction of the HNW assay would be compatible with the specificrequirements of real-time monitoring in culture, specifically:

-   -   The need to maintain viability of the culture (complex formation        with the microorganism cannot inhibit growth);    -   Ability to reliably and reproducibly disperse the magnetic        pellet once it has been formed to enable the SERS and magnetic        reagents to continue interacting with the sample;    -   Ability of SERS HNW assay signal to increase and decrease over        time in response to continuous changes in target concentration;        and    -   Ability to conduct the HNW assay on large volumes such as are        typically used in blood culture and industrial applications, as        one would have initially expected that the reagent volume        requirements would have been cost prohibitive and/or that one        would be unable to form a pellet that was representative of the        entire volume. (Any reasonable-sized magnetic field would be        expected to only pull magnetic particles from the local        micro-environment.)

An HNW assay according to an embodiment of the invention can be used todetect pathogens such as E. coli, Listeria, Salmonella, etc. growing infood or environmental samples. Since the presence of even a singledamaged organism is significant, samples are typically cultured in orderto recover and selectively grow the pathogen to a detectable level.Because the initial sample may have a range of pathogen concentrations,varying levels of damage to the pathogen, and/or highly variablecompeting background microorganisms, the required culture time to reachthe limit of detection for any given analytical method can vary wildly.For this reason, detection protocols are typically formulated for “worstcase” scenarios i.e. the length of culture time is chosen to ensure thatthe single damaged pathogen is grown to a detectable level. Detectionand identification of the pathogen (e.g., by immunoassay or PCR) is thenperformed at the completion of culture. Since the initial load ofpathogen in any given sample cannot be known a priori, all samples aresubjected to this long culture protocol to ensure that no pathogens aremissed. However, it is likely that many samples would have yieldedpositive detection and identification after shorter culture protocols,providing earlier notification to the tester that there is a problemwith the sample. The combination of the SERS-based HNW assay withculture allows real-time monitoring of the pathogen load in the samplethroughout the culture, providing the significant advantage that sampleswith higher pathogen loads are detected as early as possible in theculture protocol.

II. Systems, Methods, and Devices for the Identification ofMicroorganisms in a Microbiological Culture Sample

Embodiments of the present invention are directed to methods, systems,and devices for detecting and identifying microorganisms in a culturesample. With reference to FIG. 1, the process generally includesproviding a plurality of indicator particles, binding members, andmagnetic capture particles in a vessel and adding a sample thatpotentially includes one or more microorganisms. The vessel may alsoinclude culture or growth media to aid in selectivity or additionalgrowth of microorganisms. The sample is then incubated and agitated fora predetermined period of time. At selected time points or on apredetermined schedule over the course of incubation, a magnetic fieldis applied to the vessel so as to form a pellet. The pellet is theninterrogated with a light source to produce a detectable signal (e.g., aSERS spectrum) that is detected and analyzed. The pellet may then bedispersed and the process repeated at the next determined time point.

FIG. 2 shows one embodiment of the methodology and devices that may beused to detect and identify microorganisms in a culture sample. In thisregard, FIG. 2 illustrates that a desired volume of an environmentalsample (e.g., about 1 L or less), a food sample (e.g., about 25 g to 375g resulting in a volume of about 250 mL to 3 L), or a clinical sample(e.g., about 100 mL or less) is obtained and placed in an enrichmentvessel. In this instance, the enrichment vessel is configured tofacilitate analysis of Salmonella or Listeria assays. The enrichmentvessel is incubated for a predetermined period of time, after which apredetermined amount of sample is transferred to a detection vial in abiocontained manner, which will be explained in further detail below.The detection vial is then placed in a real-time SERS system for furtherincubation and automated analysis using SERS technology, which is alsodiscussed in further detail below.

According to one embodiment, the SERS system is configured toaccommodate a plurality of detection vials and thereby provide a highthroughput system. The SERS system may also be configured to facilitatean automated analysis of a plurality of different assays. For example,the SERS system may include dedicated zones for handling and analysis ofeach assay.

The systems and methods according to the embodiments of the inventionprovide real-time monitoring of microorganism growth in microbiologicalculture samples. FIG. 3 shows an embodiment of intermittent monitoringof microorganism growth or an endpoint embodiment. In this embodiment,SERS HNW reagents 1 are added to the vessel 2 where the culture occurs.The media 3 and sample 4 are added to the vessel 2, and the vessel 2 isplaced into an incubator 5 so that the microorganism (e.g. bacteria,yeast, or cells) is allowed to grow. At user selected time points(either during the culture or at the end of a culture period) the vesselis removed from the incubator 5 and placed in a SERS reader 6, which(after appropriate mixing of the sample) forms a magnetic pellet andreads the Raman signal. The vessel can then be reinserted into theincubator 5 to allow further growth time, if no Raman signal isdetected.

FIG. 4 shows an alternate embodiment in which the SERS signal iscontinuously monitored during bacterial growth. In this embodiment, theincubator and SERS reader are integrated into a single instrument 7which, at prescribed time points, forms the magnetic pellet, reads theSERS signal, and disperses the reagents without need for userintervention.

A. Enrichment Vessel and Detection Vial

Microbiological culture bottles, tubes, syringes, vials, vessels, andthe like (e.g., enrichment vessels and, detection vials) suitable foruse with the presently disclosed methods, systems, and devices can, insome embodiments, be made of glass or plastic. In some applications, amultilayered plastic is desirable to control gas permeability. In thoseembodiments wherein the microbiological culture vessel is made ofmultilayered plastic, the bottle may be injection or blow molded andhave inner and outer layers of polyester, polypropylene, polyethylene,polyvinyl chloride, polycarbonate, polyethylene terephthalate (PET),cyclic olefin copolymer (COC), or any copolymer or mixture thereofseparated by an intermediate layer of nylon, ethylene vinyl alcohol(EVOH), polyethylene vinyl alcohol, or copolymers or mixtures thereof.However, it is understood that the vessel may not be multilayered inother embodiments and formed using similar techniques (e.g., injectionor blow molding). In some applications, the vessel components may betreated with surface coating or chemical methods to controlvessel/sample interactions or physical properties. In some embodiments,the vessel can be transparent to visible radiation, although, inparticular embodiments, such transparency is not required. Additionally,in some embodiments, the presently disclosed vessels can be adaptable tosterilization. Further, in some embodiments, the vessel is suitable foraerobic or anaerobic culture. In one embodiment, the vessel is gaspermeable. In addition, the vessel may include a constant wall thicknessalong its length which may enhance pelleting and optical analysis.

FIGS. 5A-5E and 7 depict an enrichment vessel 50 according to oneembodiment of the present invention. Optionally, the enrichment vessel50 may hold dried or liquid culture media. The enrichment vesselgenerally includes a lid 52, a basket 54, needle assemblies 56, and acontainer 58. The lid 52 is engaged with the basket 54 and is configuredto engage and seal the container 58 in a fluid-tight connection, such asusing a threaded or snap-fit attachment. In one example, the lid 52 maybe threaded onto the container 58 but would include one or more back-offfeatures to prevent unscrewing of the lid without the additionaldisengagement of the back-off feature (e.g., press down and rotate thelid for removal). Thus, the lid 52, needle assemblies 56, and basket 54may be coupled together so as to be able to engage and disengage thecontainer 58 as a unit. For example, the lid 52 and basket 54 may becoupled together in a snap fit or using other suitable techniques suchas adhesives, heat staking, or fasteners. In this regard, FIG. 9Cillustrates that the basket 54 may include fastener holes 60 forengagement with fasteners 62 to secure the lid and basket together (seealso FIG. 5A). FIG. 8 shows the bottom of the lid including a pluralityof holes 65 that align with respective holes 60 on the basket (see FIG.9C) for receiving the fasteners 62 therethrough. Likewise, the needleassemblies 56 may be attached to the lid 52 using similar securementtechniques, such as a force fit, threaded engagement, or adhesives. Thecontainer 58 is configured to hold a desired amount of sample thereinand thus, may be various sizes and shapes as needed. For example, FIGS.5A-5C, 7, and 100 illustrate exemplary shapes of a container 58. In oneembodiment, the basket 54 and container 58 may be transparent ortranslucent to facilitate visibility within the container and inparticular, visibility of the sample within the reservoirs 64, 66. Inaddition, FIG. 100 illustrates that the container 58 may include one ormore volume lines 59 for visualizing the amount of sample contained inthe container. FIG. 7 also illustrates that the vessel 50 may include agasket 68 or other sealing member used to ensure a fluid-tightconnection between the lid 52 and the container 58.

The enrichment vessel 50 includes a pair of needle assemblies 56 andreservoirs 64, 66. However, it is understood that there may one or moreneedle assemblies 56 and reservoirs 64, 66 in alternative embodiments.In the illustrated embodiment, one needle assembly 56 and reservoir 64or 66 is configured for use with a particular type of assay (e.g.,Salmonella or Listeria). Because different microorganisms are culturedusing different media and sample sizes, the enrichment vesselfacilitates use of a single basket for different assays.

The basket 54 is shown in more detail in FIGS. 9A-9C. The basket 54includes a pair of reservoirs 64, 66, with each reservoir configured tohold a predetermined sample volume. As shown, the reservoirs 64, 66 arespaced away from the bottom of the container 58, wherein this space isconfigured to hold a desired sample. In this regard, the first reservoir66 is configured to hold a larger volume than the second reservoir 64.In one specific embodiment, the first reservoir 66 is configured to holdabout 5 mL and the second reservoir 64 is configured to hold about 100μL. As shown, the reservoirs 64, 66 may be shaped to facilitate meteringof the sample as well as alignment with a respective needle assembly 56.For example, FIGS. 5A-5C and 6 illustrate that each needle 70 isinserted within a reservoir 64, 66 and to the lowest position therein toensure that substantially all of the metered sample is removed. Thus,the length of the needle 70 may be adjusted depending on the size of thereservoir, as the needle extending within the first reservoir 66 islonger than the needle extending within the second reservoir 64. Theshape of the reservoir 64, 66 may be any shape that is suitable toretain the desired amount of sample. For example, FIGS. 5A, 5B, 5C, 5E,and 9B show that the second reservoir 64 has a generally conical shape,while the first reservoir 66 has surfaces that extend along the needleand taper towards the base of the needle.

FIG. 9C particularly illustrates that the basket 54 includes a number ofholes 72, 74 defined therethrough. Typically, the amount of sample inthe container 58 would be below the holes 72 when the container is in anupright position, but would be at least below the entrance to eachreservoir 64, 66 so that a desired volume can be metered. Holes 72 maybe defined within the basket adjacent the reservoirs, while holes 74 maybe defined through an upper surface 76 of the basket adjacent alid-engaging portion 78. The holes 72 located adjacent the reservoirs64, 66 are configured to drain excess sample within a respectivereservoir. Namely, when the enrichment vessel 50 is tilted from anupright horizontal position to fill one of the reservoirs 64, 66,returning the vessel to the upright position results in the reservoirbeing over-filled with sample and excess sample will subsequently drainthrough the holes 72. As such, a desired volume is metered in arepeatable manner within each reservoir 64, 66. The holes 74 defined inthe upper surface 76 of the basket 54 may be used to allow sample toenter the reservoirs 64, 66 while also preventing unwanted particulatesin the sample from being transferred into the reservoirs. It isunderstood that the basket 54 may be modified depending on the amount ofsample to be metered and the type of sample, such as by modifying thesize and depth of the reservoir 64, 66, as well as the size and depth ofthe holes. In this regard, FIG. 9C illustrates that the holes 72, 74 maybe tapered in different directions from one another for aiding indraining, with the entrance to the holes 72 adjacent the reservoir beinglarger than the entrance to the holes 74 in the upper surface 76 of thebasket. The smaller hole entrance may be used to filter any undesirableparticles from entering the reservoirs. In addition, FIGS. 106-109illustrate an embodiment where the basket 54 includes holes 72, 74 thatare approximately the same size. Moreover, the basket 54 may alsoinclude a rib 75 or other raised surface that is configured to aid indraining of fluid through the basket. In particular, the rib 75 may beat the center of the basket 54 to facilitate venting during filteringand draining by offering a surface capable of draining excess fluidabove the basket with different draining characteristics than theremainder of holes in the upper surface 76 of the basket. FIGS. 106 and108 illustrate an embodiment wherein a bottom surface of the reservoirs64, 66 may include one or more protrusions 119 which aid draining bywicking fluid from the reservoir and allowing fluid from multiple drainholes 72 to coalesce.

As shown in FIG. 9B, each reservoir 64, 66 is separated from an uppersurface 76 of the basket with a respective head space 80, 82. The headspaces 80, 82 allow the sample to readily enter a respective reservoir64, 66 when the container is tilted. Thus, when the container 58 istilted, sample enters through the holes 74 defined in the upper surface76 of the basket 54, into the head space 80 or 82, and enter thereservoir 64 or 66. When the container 58 is returned to an uprightposition, the reservoir 64 or 66 is overfilled due to excess samplelocated in the head space 80 or 82, wherein the excess sample thendrains through the holes 72 defined adjacent the reservoir and back intothe container. As shown in FIG. 9B, the holes 72 defined adjacent thereservoirs 64, 66 are located below the opening leading into thereservoir to facilitate draining and metering the desired amount ofsample.

Each reservoir 64, 66 is aligned with a respective needle assembly 56 asshown in FIGS. 5A-5C and 6. In one embodiment the lid 52 includes alid-engaging portion 78, wherein the lid engaging portion is configuredto couple the basket 54 and lid together as discussed above. Thelid-engaging portion 78 and basket 54 have a smaller outer diameter thanthe inner diameter of the opening of the container 58 so as to beconfigured to be inserted within the container. The lid-engaging portion78 may also include openings for receiving respective needle assemblies56 that extend into the reservoirs 64, 66. The needles 70 are locatedwithin the lid-engaging portion 78 so that the needles are configured toengage a detection vial as discussed in further detail below. In thisregard, the lid-engaging portion 78 includes a conical or taperedsurface 105 opposite a respective opening 102, 104 that is configured toreceive and engage with a needle assembly 56. The needles 70 furtherextend through respective openings defined in the bottom of the conicalsurface 105 into the head space 80, 82 and within a respective reservoir64, 66 (see FIGS. 5A-5C and 6). FIG. 6 illustrates that each needleassembly 56 may be engaged with the lid engaging portion 78 such thatthe needles 70 extend proximate the reservoirs 64, 66. FIG. 6illustrates that the lid 52 may also include a vent 86 defined thereinfor allowing any nonhazardous, gaseous byproducts to escape from thecontainer to prevent pressure build up during culture. FIGS. 102-103 and105 illustrate an alternative embodiment where a vent post 125 extendsfrom a bottom surface of the lid 52. The vent post 125 defines anopening therethrough for receiving and engaging a filter for filteringany gaseous byproducts exiting the container 58. The vent post 125aligns with vent 86. In this regard, the vent post 125 is configured todirect nonhazardous, gaseous byproducts through the opening in the ventpost and through the vent 86 defined in an upper surface of the lid 52.

FIGS. 102-103 and 105 also illustrate that the lid 52 may furtherinclude an engagement post 127 extending outwardly from a bottom surfaceof the lid. The engagement post 127 is configured to align with andengage a corresponding engagement post 129 extending outwardly from anupper surface of the basket 54, as shown in FIGS. 106, 107, and 109. Asillustrated, the engagement post 127 has a smaller diameter thanengagement post 129, although the relative sizes of the posts may bereversed if desired. In addition, the engagement portion 78 shown inFIGS. 103-105 is configured to engage a corresponding engagement portion121 defined on an upper surface of the basket 54 (see FIGS. 106, 107,and 109). In particular, the engagement portion 78 may be sized andconfigured to overlie and encircle the engagement portion 121. The outerperiphery of the engagement 121 surface may define a plurality of ribs123. When the engagement surfaces 78 and 121 are brought into engagementwith one another (e.g., by sliding and/or rotating with respect to oneanother), the ribs may be configured to compress the ribs 123. Thecompression may be sufficient to create a friction fit between the lid52 and the basket 54. In one embodiment, the ribs 123 may be crushed orotherwise deformed to create a friction fit.

Each needle assembly 56 is configured to engage a respective detectionvial 100. The detection vial 100 may include a particular capconfiguration for mating with a respective opening 102, 104 defined inthe lid 52. Thus, each cap may be associated with a specific type ofsample so that the risk of using the wrong media for a microorganism isminimized. For example, the lid 52 may include a keyed opening 104 thatonly allows mating with the cap of the detection vial when the cap isoriented to engage the keyways 110 (see FIG. 6). FIG. 101 shows analternative embodiment of a lid 52 where keyways 110 are defined alongthe length of the opening 104, including along conical surface 105. Thekeyways 110 may be defined on the inner surface of the opening, as shownin FIG. 6, or on both the inner and outer surfaces of the opening asshown in FIGS. 101-103.

FIGS. 10A and 10B illustrate an exemplary embodiment of a cap 106suitable for use with a detection vial. In this regard, the cap 106includes a plurality of ribs 108 that are configured to engagerespective keyways 110 defined in the opening 104 of the lid 52 (seeFIG. 5D). Thus, in order for the cap 106 to be inserted within opening104, ribs 108 would need to be radially aligned within the keyways 110.In addition, the cap 106 includes a plurality of engagement features 112that are configured to engage the detection vial 100 in a snap fit. Thesnap connection may minimize ovalization of the detection vial 100, aswell as dislodgement of the cap 106 during handling. It is understoodthat the cap 106 and detection vial 100 may be secured together usingother suitable techniques, such as a threaded or crimped sleeve/capengagement, adhesives, ultrasonic welding, and/or heat staking. FIG. 13Aillustrates the cap 106 engaged with a detection vial 100, according toone embodiment of the present invention.

As mentioned above, the cap 106 may have different configurations fordifferent assays so that the risk of using the incorrect detection vial100 is eliminated. For instance, FIGS. 11A and 11B illustrate analternative cap 114 configuration, while FIG. 14A shows the cap 114engaged with a detection vial 100. As illustrated, each cap 106, 114 mayinclude a plurality of ribs 108 and engagement features 112. The cap 114may be configured to be received within a respective opening 102,although the opening need not include corresponding keyways. Thus, thecap 114 may be received within the opening 102 regardless of its radialorientation, but the cap 114 would be incapable of being inserted withinopening 104. Thus, the ribs 108 of cap 106 may prevent access to theopening 102 in the lid 52 just as the outer diameter of cap 114 mayprevent access to opening 104.

FIGS. 12, 13B, and 14B illustrate additional features of the detectionvial 100 and cap 114 according to one embodiment of the presentinvention. Namely, the cap 114 includes a stopper 116 and an absorbentpad 118 disposed between the cap and the stopper. The absorbent pad 118may be used to absorb any sample that exits the detection vial 100 aftertransferring the sample from the enrichment vessel 50 into the detectionvial thereby minimizing exposure to the technician or environment. Thecap 114 may also have a finger stand-off 117 to prevent accidentalcontact of a potentially wetted pad. Moreover, the stopper 116 may beany suitable material that is configured to create a fluid-tightconnection with the detection vial 100 (i.e., liquid and gas), as wellas to be pierced by a needle to reseal to a fluid tight connection afterbeing pierced by a needle and to engage the detection vial. Forinstance, the stopper 116 may be a suitable rubber or elastomericmaterial. FIG. 12 also illustrates the engagement between the engagementfeatures 112 and the protrusion 115 of the detection vial 100. Thus, thecaps 106, 114 may engage with the detection vial 100 in a snap-fit,which would prevent unintentional removal or dislodgement of the cap.The shape of the protrusion 115 can be any shape that would allow for aretentive snap fit with the engagement features 112.

The detection vials 100 may include the reagents and optionally a media,such as for example a specific growth media, depending on themicroorganism that is being tested in the sample. The reagents and mediamay be present in the detection vial in a dried (e.g., dehydrated)format or in a wet (e.g., hydrated) format. For example, the media andreagent may be dried. The detection vials 100 may also hold a vacuumwhen the stopper 116 is engaged therewith. Thus, when the detection vial100 is inserted within a respective opening 102, 104 in the lid 52, thestopper 116 and absorbent pad 118 are pierced by the needle 70, and thesample within the reservoir 64 or 66 is pulled through the needle andinto the detection vial (see FIGS. 15-17). In one example, the portionof the needle 70 extending into the opening 102 or 104 may include aprotective sleeve 120 that is configured to be compressed as thedetection vial 100 is pushed downwardly and into engagement with theneedle. When the protective sleeve 120 is compressed, the needle 70 isexposed and penetrates the stopper 116, allowing the vacuum to pull theportion of the sample contained within the reservoir 64 or 66. Aftercompletion of the fluid transfer and removal of the detection vial 100,the protective sleeve 120 returns to its original shape covering theneedle 70. As such, the transfer of sample between the enrichment vessel50 and the detection vial 100 occurs in a biocontained manner. Thevacuum within each detection vial 100 may be any amount sufficient topull a desired amount of sample from the reservoir (e.g., 8-10 mL drawcapacity for 5 mL sample). Any further excess vacuum in the detectionvial 100 is exhausted by air following the fluid from the reservoir 64or 66.

The detection vial 100 may be provided with reagents, with or withoutculture or growth media, stopper 116, pad 118, and cap 106 or 114 withvacuum or without vacuum, depending on the end-user (e.g., outsourceduse versus in-house use). In this vein, the detection vial 100 may beassembled only to the stopper 116 for retention of reagents only, whilethe cap 106 or 114 is supplied separately for users who need to accessthe interior of the detection vial. Alternatively, the detection vial100 can be pre-assembled with a stopper 116, pad 118, and cap 106, 114combination as shown in FIG. 12. Furthermore, the media and reagentamount is determined by the detection vial 100, not the initialenrichment volume, according to one embodiment.

As such, the configuration of the enrichment vessel 50 and detectionvial 100 enable the sample to be contained and transferred in abiocontained manner, thereby limiting exposure to the technician orfacility. The enrichment vessel 50 also facilitates accurate metering ofa desired volume of sample, while also being configured to accommodate aplurality of types of samples. For example, this may be particularlyuseful for Salmonella and Listeria, where different assays, media, andamount of sample are utilized. The enrichment vessel 50 and detectionvial 100 are also configured to reduce the risk that the incorrect vialwill be used for testing by incorporating mating features between theenrichment vessel and the detection vial.

FIGS. 86 and 88 illustrate another embodiment of an enrichment vessel125. As before, the vessel 125 includes a lid 126 engaged with acontainer 128 in a fluid-tight manner. As shown, the enrichment vessel125 includes a longitudinal syringe support 130 extending from the lid126 and into the container 128. The syringe support 130 may be attachedto the lid 126 or may be integrally formed therewith. The syringesupport 130 includes an opening 132 configured to receive a syringe 134therein, and is generally shaped in a mating relationship with thesyringe 134. Engaged at the base of the syringe support 130 is a needleassembly including a hub 133 and a needle 135, wherein the hub isengaged with the syringe support, and the needle extends within thecontainer 128 and is configured to draw sample therethrough. The needle135 may include a compressible cover 141 that extends over the portionof the needle within the syringe 134. As also discussed above, the lid126 may include a vent 131 for allowing nonhazardous, gaseous byproductsto escape from the container.

FIG. 87 illustrates one embodiment of a syringe 134 that generallyincludes a handle 136, a plunger rod 137 coupled to the handle, a cap138, a plunger 139 coupled to the end of the plunger rod, a septum 140,and a seal 146. FIG. 87 further illustrates that the syringe 134 isconfigured to engage the opening 132, such as with a twist-lockinterface 142, for supporting the pull force applied while drawing thesample out of the container 128. Thus, the plunger 137 is longitudinallydisplaceable within the syringe 134. The septum 140 is configured to bepierced by a needle and reseal upon removal of the needle. In otherembodiments, the septum 140 also includes an absorbent pad and astand-off feature to prevent contamination to the user due to any fluidthat may escape through the septum. The seal 146 is engaged with thesyringe 134 and cap 138 to create a fluid-tight connection. The syringe134 may also include a first larger bore 149 disposed within the opening132 and a neck region 157 including a second smaller bore 151. As shownin FIGS. 87 and 88, an extension 153 extends from the neck region 157and into the larger bore 149 such that a portion of the smaller bore 151extends within the larger bore 149. There may be one or more slots oropenings 155 defined between the extension 153 and the base of the neckregion 157, as discussed in greater detail below.

FIGS. 89A-89C illustrate the progression of a start position, a “soft”stop, and a “hard” stop when removing sample from the container 128 andinto the syringe 134. As shown in FIG. 89A, when the syringe 134 isengaged with the syringe support 130, the plunger 139 is positionedadjacent to the needle 135 in the start position and is configured tocompress the cover 141 in order to allow fluid communication between thecontainer 128 and the syringe 134 via the needle. FIG. 88 furtherillustrates that the needle 135 is configured to penetrate the septum140 when the syringe 134 is engaged with the syringe support 130. As theplunger rod 137 is pulled outwardly from the syringe 134, a portion 144of the sample is pulled through the needle 135 and into the bore 151,and a first engagement feature 145 on the plunger rod engages the seal146 to stop further withdrawal thereof. In this manner, the sample iswithdrawn at a desired rate whereby the fluid is able to catch up withthe vacuum so that underdrawing the sample is prevented.

In FIG. 89C, the plunger rod 137 is withdrawn further from the syringe134 whereby a second engagement feature 147 on the plunger rod 137engages the seal 146 to prevent further withdrawal of the plunger rod.In addition, the first engagement feature 145 is engaged within a pocket148 defined in the seal 146 that prevents the plunger rod 137 from beingdisplaced further out of the syringe 134. As shown in FIG. 89C, theplunger 139 may be disposed within the extension 153 of the syringe 134such that no further vacuum may be pulled due to the plunger rod 137 andplunger 139 being positioned so that the bore 151 is no longer closed.That is, when the plunger 139 is no longer covering the openings 155,the bores 149 and 151 are in fluid communication with one another suchthat a vacuum is no longer being pulled. Moreover, the engagement of theplunger rod 137 and the seal 146 prevents the plunger rod from beingpulled back into the syringe 134, while also preventing a user fromfurther withdrawing the plunger and risking exposure to the sample.

FIGS. 90A and 90B illustrate another embodiment of a plunger rod 137.The plunger rod 137 includes longitudinal ribs 159 that are configuredto slide within slots defined within the cap 138. Moreover, FIG. 90Bshows that the plunger 137 is able to be twisted to disengage the ribs159 from the slots and engage the cap 138 to prevent the plunger fromreturning into the syringe 134.

FIGS. 91 and 92 illustrate alternative plunger rods and plungers thatmay be used for withdrawing different volumes from the container 128. Inthis regard, FIG. 91 corresponds to that described in connection withFIGS. 87, 88, and 89A-C. Thus, the plunger 139 is suitable forwithdrawing smaller volumes into the smaller bore 151 (e.g., about 125μL). Accordingly, the size of the plunger and length of the plunger rodmay be varied as needed. In another embodiment, FIG. 92 shows a plunger161 suitable for withdrawing sample into the larger bore 149 (e.g.,about 5 mL). Thus, the syringe 134 is suitable for use with differentplungers for withdrawing different volumes of sample, which is usefulwhen testing for different microorganisms (e.g., Listeria andSalmonella). Moreover, the user may receive the syringe 134 andplunger/plunger rod pre-assembled, or the user may be able to add inreagents, reconstitution fluid, etc. and then assemble theplunger/plunger rod to the syringe.

A method for the detection and identification of one or moremicroorganisms in a microbiological culture sample according to anembodiment of the invention can be performed in a microbiologicalculture vessel. A microbiological culture vessel can have disposedtherein one or more indicator particles and one or more magnetic captureparticles each having associated therewith one or more binding members,e.g., an antibody, having an affinity for the one or more microorganismsunder test. The indicator particles and magnetic capture particles canbe disposed in the microbiological culture vessel prior to, concurrentwith, or subsequent to disposing therein a clinical or industrial samplesuspected of containing the one or more microorganisms under test. Theculture growth media can be disposed in the microbiological culturevessel prior to, concurrent with, or subsequent to addition of theclinical or industrial sample. Once the indicator particles, magneticcapture particles, culture media, and clinical or industrial sample havebeen introduced into the culture vessel, the culture vessel is thenagitated either continuously or intermittently in order to mix theindicator particles and magnetic capture particles with the combinedsample and culture medium. In preferred embodiments described herein theagitation profile (e.g., speed and/or displacement) may be varied atdifferent stages of the culture or read cycle. When present in theclinical or industrial sample, the one or more microorganisms under testcan bind with the one or more binding members associated with theindicator particles and magnetic capture particles to form a magneticcapture particle-microorganism-indicator particle complex.

Where the indicator particle is SERS-active, FIG. 18 shows an example ofa magnetic capture particle-microorganism-SERS-active indicator particlecomplex within a culture vessel 2. The SERS-active indicator particle10, has associated therewith one or more specific binding members 11having an affinity for one or more microorganisms 12 under test. Amagnetic capture particle 13 also has associated therewith one or morespecific binding members 14 having an affinity for the one or moremicroorganisms 12 under test. Magnetic capture particles 13 can bind toone or more microorganisms 12, which also can be bound to SERS-activeparticle 10, to form the magnetic captureparticle-microorganism-SERS-active particle complex, which is alsoreferred to herein as a sandwich complex, wherein the microorganism isbound simultaneously by more than one specific binding member. In thisparticular sandwich complex, at least one specific binding member 14 isattached to a magnetic capture particle 13 and at least one otherspecific binding member 11 is attached to a SERS-active indicatorparticle 10. Thus, the microorganism 12 is “sandwiched” between themagnetic capture particle 13 and the SERS-active indicator particle 10.

A magnetic field is applied to the sample via a magnet 15 to attract themagnetic capture particles 13 in order to localize the magnetic captureparticle-microorganism-SERS-active indicator particle complexes into apellet within the measurement zone 9 inside of the culture vessel 2 fordetecting the SERS signal. Radiation from light source 16 can then bedirected at the pellet and the SERS signal can be detected by Ramandetector 17. Light source 16 and detector 17 are used to induce andmeasure, respectively, the Raman signature produced by SERS-activeindicator particle 10. The localization of the magnetic captureparticle-microorganism-SERS-active indicator particle complexes providesa SERS signal, the intensity of which is reflective of microorganismconcentration, by localizing the SERS-active indicator particles thatare bound to magnetic particles in the detection zone, therebysegregating them from the unbound SERS-active indicator particlesremaining in solution.

In some embodiments, the measurement zone can be located along an innersurface of a microbiological culture bottle or vessel. For example, withrespect to a bottle, the measurement zone can be located along an innersurface within or adjacent to the bottle neck; an inner surfacecomprising the bottle mid-section; or an inner surface along the base ofthe bottle adjacent to, for example, a separate sensor, e.g., afluorescence-based sensor or a colorimetric-based sensor, or inembodiments in which a separate sensor is not present, along an innersurface of the base, i.e., the bottom, of the microbiological culturebottle. In one preferred embodiment, the measurement zone is locatedalong an inner surface generally at the mid-section of the culturebottle or vessel. Thus, the measurement zone may be located at or closerto the center of the bottle or vessel than the ends of the bottle orvessel (e.g., within the middle 50% of the vessel).

The detection and/or identification of the one or more microorganisms ofinterest is accomplished only when the microorganism(s) is/are bound inthe pellet as part of a binding member-microorganism-indicator particlecomplex. That is, no signal is generated when the one or moremicroorganisms are not present in the microbiological culture sample or,if present, the microorganism does not have an epitope recognized by thebinding member associated with the indicator particle. Under suchcircumstances, the indicator particles are not substantially present inthe measurement zone.

If no significant SERS signal is observed upon application of a magneticfield and optical interrogation of the pellet, the magnetic particlespulled into the pellet may be dispersed back into solution in order tocontinue interacting with the sample. If a microorganism is presentbelow the limit of detection of the technology, then the microorganismconcentration can increase over time as the microorganism grows in theculture media so that the SERS signal is ultimately detected in themeasurement zone upon future application of the magnetic field. Inessence, a magnetic pellet is formed, optically interrogated, dispersed,allowed to interact with the sample, and then reformed at a specifiedfrequency until either a signal is observed from the bindingmember-microorganism-indicator particle complex or the sample isdetermined to be negative for the microorganism of interest. Agitationof the culture vessel at various stages throughout this process may playa critical role. Agitation serves a variety of purposes. First, itensures mixing of the SERS and magnetic particles with the sample andculture media allowing the formation of bindingmember-microorganism-indicator particle complexes. Second, it enablesthe dispersion of the magnetic particles back into solution once thepellet is formed. Third, in a preferred embodiment, agitation can occurwhile the magnetic field is applied. Agitation during application of themagnetic field brings fluid from various spatial points within theculture vessel into the region of the localized magnetic field, ensuringthat magnetic particles are collected from regions of the sample outsideof the localized magnetic field. Finally, in samples containingparticulates (e.g. resins, charcoal, or calcium carbonate), agitationprior to and during pelleting can limit the number of these particulatesfrom settling into the detection region and interfering with the opticalsignal. Different agitation rates and profiles may be optimum for eachof these different functions.

For example, different agitation rates (i.e., frequency) and “throw”(i.e., vial displacement along an axis) may be used in different phasesof a measurement cycle. In one exemplary embodiment, a measurement cyclemay include mixing, pre-pellet dispersion, pelleting, reading, anddispersion, with each phase having a particular agitation rate andthrow. In this regard, mixing includes the phase where agitation occursduring incubation, while pre-pellet dispersion occurs after mixing andprior to pelleting. Pelleting proceeds after pre-pellet dispersion andis followed by the reading phase. The reading phase corresponds to theinterrogation of the vials by the read head, while the dispersion phaseis provided for the pellet to be redispersed within the vial. There mayor may not be delays between phases. In one embodiment, the agitationrate and throw for the phases may range from about 0 to 3 Hz and about 0to 100 mm, respectively. For instance, the following agitation rates andthrows may be used according to embodiments of the present invention:mixing—about 0.5 to 1.5 Hz and 25 to 75 mm; pre-pellet dispersion—about1 to 2 Hz and 25 to 75 mm; pelleting—about 0.5 to 2 Hz and 25 to 75 mm;reading—0 Hz and 0 mm; and dispersion—about 1 to 2 Hz and about 25 to 75mm. Moreover, the particular time period for each phase may also bevaried. For example, the mixing phase may be significantly longer (e.g.,about 5 to 60 min) than the pre-pellet dispersion, pelleting, reading,and dispersion phases (e.g., about 5 to 120 seconds per phase).

FIG. 23 shows one example of the time-dependent SERS signal intensity ofbinding member-microorganism-indicator particle complexes captured bymagnetic capture particles for Salmonella. Generally, the beginning ofthe upslope of the SERS signal may be indicative of the presence of themicroorganism. In this regard, after about 6 hours of culture time, thepresence of microorganism may be indicated, while the peak at about 9hours indicates a higher concentration of microorganisms. However, asalso shown in FIG. 23, on the downslope of the SERS signal,microorganisms may continue to be present, such as at about 12 hours. Inthis instance, the downslope may also signify the presence of amicroorganism, which may provide a useful means of identifyingpositivity in certain cases, such as when a large number ofmicroorganisms are present at the beginning of incubation. As such,either an upslope or downslope in the SERS signal may be indicative ofthe presence of a microorganism in the culture sample. In this regard,readings may be taken periodically over time during the incubationperiod in order to identify such changes in the SERS signal.

B. Indicator Particles

“Indicator particles”, as used herein, may be any particle that iscapable of producing a signal that can be detected directly in theculture sample without removing the sample, such as for performing washsteps. For example, the indicator particles may produce any opticalsignal (e.g., fluorescence or Raman or an optical image) wheninterrogated (e.g., with a light source). Examples of indicatorparticles include SERS-active particles, quantum dots, near-infraredfluorophores, or near-infrared fluorescent particles.

“Surface-enhanced Raman scattering” or “SERS” refers to the phenomenonthat occurs when the Raman scattering signal, or intensity, is enhancedwhen a Raman-active molecule is adsorbed on or in close proximity to,e.g., within about 50 Å of, the surface of certain metals (e.g., gold orsilver). Under such circumstances, the intensity of the Raman signalarising from the Raman-active molecule can be enhanced.“Surface-enhanced resonance Raman scattering” or “SERRS” refers to anincreased SERS signal that occurs when the reporter molecule in closeproximity to a SERS-active nanoparticle surface is in resonance with theexcitation wavelength. “Raman scattering” generally refers to theinelastic scattering of a photon incident on a molecule. Photons thatare inelastically scattered have an optical frequency (vi), which isdifferent than the frequency of the incident light (v0). The differencein energy (ΔE) between the incident light and the inelasticallyscattered light can be represented as (ΔE)=h|v0−vi|, wherein h isPlanck's constant, and corresponds to energies that are absorbed by themolecule. The incident radiation can be of any frequency v0, buttypically is monochromatic radiation in the visible or near-infraredspectral region. The absolute difference |v0−vi| is an infrared, e.g.,vibrational, frequency. The frequency v1 of the “Raman scattered”radiation can be greater than or less than v0, but the amount of lightwith frequency v1<v0 (Stokes radiation) is greater than that withfrequency v1>v0 (anti-Stokes radiation).

As used herein, the term “radiation” refers to energy in the form ofelectromagnetic radiation that can induce surface-enhanced Ramanscattering in a sample under test, e.g., a sample comprising aSERS-active nanoparticle having one or more SERS-active reportermolecules associated therewith. More particularly, the term “radiation”refers to energy in the form of electromagnetic radiation that causesthe surface of a nanoparticle to induce, emit, support, or otherwisecause light scattering, e.g., Raman scattering, in a reporter moleculeproximate to the nanoparticle surface.

As used herein, a “reporter molecule” refers to any molecule or chemicalcompound that is capable of producing a Raman spectrum when it isilluminated with radiation of a proper wavelength. A “reporter molecule”also can be referred herein as a “label,” a “dye,” a “Raman-activemolecule,” or “SERS-active molecule,” each of which can be usedinterchangeably.

One of ordinary skill in the art would appreciate that a variety ofmolecules can act as SERS reporter molecules. For example, somefluorescent dye molecules also can be used as SERS reporter molecules.See, e.g., U.S. patent application Ser. No. 12/134,594 to Thomas et al.,filed Jun. 6, 2008, and PCT International Patent Application No.PCT/US2008/066023 to Thomas et al., filed Jun. 6, 2008, each of which isincorporated by reference in its entirety. Generally, molecules suitablefor use as SERS reporter molecules can be a small molecule, a largemolecule, or a complex molecule, although the molecule does not need tobe complex to act as a SERS reporter molecule. SERS reporter molecules,in some embodiments, can have at least one aromatic ring. Further,without wishing to be bound to any one particular theory, a change inpolarizability of a bond is required for Raman activity. Also, symmetricmolecules tend to exhibit specific and strong Raman signals.Advantageously, a reporter molecule exhibits a high Raman scatteringcross section and a well-characterized spectral signature.

A SERS-active nanoparticle, as referred to herein, includes ananoparticle having a surface that induces, causes, or otherwisesupports surface-enhanced Raman light scattering (SERS) orsurface-enhanced resonance Raman light scattering (SERRS). A number ofsurfaces are capable of producing a SERS signal, including roughenedsurfaces, textured surfaces, and other surfaces, including smoothsurfaces.

A SERS-active indicator particle suitable for use with the presentlydisclosed assays includes a core, which induces the Raman effect, andcan further include one or more layers and types of SERS-activematerials located on the outer surface of the core, and optionally anencapsulant which partially or fully encapsulates the core or the SERSactive materials.

FIGS. 19-21 show various examples of SERS-active indicator particles.FIG. 19 shows a SERS-active indicator particle 20 with a singleSERS-active nanoparticle 21 as a core, having a reporter molecule 22located on the outer surface of the nanoparticle core and a layer ofsilica 23 fully encapsulating the core and reporter molecule. SuchSERS-active indicator particles are described in U.S. Pat. No. 6,514,767to Natan, which is incorporated herein by reference in its entirety.

As used herein, the term “nanoparticle,” refers to a particle having atleast one dimension in the range of about 1 nm to about 1000 nm,including any integer value between 1 nm and 1000 nm (including about 1,2, 5, 10, 20, 50, 60, 70, 80, 90, 100, 200, 500, and 1000 nm). In someembodiments, the core of the SERS-active indicator particle is ametallic nanoparticle. In some embodiments, the SERS-active indicatorparticle is a spherical particle, or substantially spherical particlehaving a diameter between about 2 nm and about 200 nm (including about2, 5, 10, 20, 50, 60, 70, 80, 90, 100, and 200 nm). In some embodiments,the SERS-active indicator particle has a diameter between about 2 nm andabout 100 nm (including about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,and 100 nm) and in some embodiments, between about 20 nm and 100 nm(including about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 nm).

SERS-active indicator particles suitable for use with the presentlydisclosed assays also can include a core comprising two or morenanoparticles. FIG. 20 shows a SERS-active indicator particle 30 with afirst SERS-active nanoparticle 31 and a second SERS-active nanoparticle32 in the core, having a reporter molecule 33 located in between thefirst 31 and second 32 SERS-active nanoparticles. Such SERS-activeindicator particles are described in U.S. Pat. No. 6,861,263 to Natan,which is incorporated herein by reference in its entirety. See also, foranother example, U.S. Patent Application Publication No. 2003/0232388 toKreimer et al., published Dec. 18, 2003, which is incorporated herein byreference in its entirety. Thus, the core a SERS-active indicatorparticle can include a single nanoparticle or can include multiplenanoparticles aggregated together. Such aggregates also can beencapsulated as further disclosed herein.

The core of a SERS-active indicator particle suitable for use with thepresently disclosed methods typically comprises at least one metal,i.e., at least one element selected from the Periodic Table of theElements that is commonly known as a metal. Suitable metals includeGroup 11 metals, such as Cu, Ag, and Au, or any other metals known bythose skilled in the art to support SERS, such as alkali metals. In someembodiments, the core nanoparticle substantially comprises a singlemetal element. For example, the preparation of gold nanoparticles isdescribed by Frens, G., Nat. Phys. Sci., 241, 20 (1972). In otherembodiments, the core nanoparticle comprises a combination of at leasttwo elements, such as an alloy, for example, a binary alloy. In someembodiments, the core nanoparticle is magnetic.

In other embodiments, the core of a SERS-active indicator particleincludes two components in which a first material forms an inner corewhich surrounded by a shell formed from a second material, such as in anAu₂S/Au core-shell particle. FIG. 21 shows such a SERS-active indicatorparticle 40 with an inner core 41 of Au₂S surrounded by an outer shell42 formed from Au as a core, having a reporter molecule layer 43 locatedon the outer surface of the core and a layer of silica 44 fullyencapsulating the core and reporter molecule layer. Au₂S/Au core-shellparticles have been reported to have widely tunable near-IR opticalresonance. See Averitt, R. D., et al., “Ultrafast optical properties ofgold nanoshells,” JOSA B, 16(10), 1824-1832 (1999). Further, Ag core/Aushell particles, such as those described by Cao, Y. W., et al.,“DNA-modified core-shell Ag/Au nanoparticles,” J. Am. Chem. Soc.,123(32), 7961-7962 (2001), or Au core/Ag shell particles, or anycore-shell combination involving SERS-active metals, can be used. Othercombinations suitable for use in core-shell particles also are suitablefor use with the presently disclosed subject matter, including Au- orAg-functionalized silica/alumina colloids, Au- or Ag-functionalized TiO₂colloids, Au nanoparticle capped-Au nanoparticles (see, e.g., Mucic, etal., “DNA-directed synthesis of binary nanoparticle network materials,”J. Am. Chem. Soc., 120(48), 12674 (1998)); Au nanoparticle-capped TiO₂colloids; and particles having a Si core with a metal shell (i.e.,“nanoshells”), such as silver-capped SiO₂ colloids or gold-capped SiO₂colloids. See, e.g., Jackson, et al., Proc. Natl. Acad. Sci. U.S.A.101(52):17930-5 (2004); see also U.S. Pat. Nos. 6,344,272 and 6,685,986to Oldenburg et al., each of which is incorporated herein by referencein its entirety. The use of such nanoshells in biosensing applicationshas been described. See U.S. Pat. No. 6,699,724 to West et al., which isincorporated herein by reference in its entirety.

Another class of nanoparticles suitable for use as a core of aSERS-active indicator particle includes nanoparticles having an internalsurface. Such nanoparticles include hollow particles and hollownanocrystals or porous or semi-porous nanoparticles. See, e.g., U.S.Pat. No. 6,913,825 to Ostafin et al., which is incorporated herein byreference in its entirety. In some embodiments, core/shell andnanoparticles having an internal surface can exhibit an improved SERSsignal.

While it is recognized that particle shape and aspect ratio can affectthe physical, optical, and electronic characteristics of nanoparticles,the specific shape, aspect ratio, or presence/absence of internalsurface area does not bear on the qualification of a particle as ananoparticle. Accordingly, nanoparticles suitable for use as a core of aSERS-active indicator particle can have a variety of shapes, sizes, andcompositions. Further, the nanoparticle core can be solid, or in someembodiments, as described immediately hereinabove, hollow. Non-limitingexamples of suitable nanoparticles for use as a core include colloidalmetal hollow or filled nanobars, magnetic, paramagnetic, conductive orinsulating nanoparticles, synthetic particles, hydrogels (colloids orbars), and the like. It will be appreciated by one of ordinary skill inthe art that nanoparticles can exist in a variety of shapes, includingbut not limited to spheroids, rods, disks, pyramids, cubes, cylinders,nanohelixes, nanosprings, nanorings, rod-shaped nanoparticles,arrow-shaped nanoparticles, teardrop-shaped nanoparticles,tetrapod-shaped nanoparticles, prism-shaped nanoparticles, and aplurality of other geometric and non-geometric shapes.

Further, nanoparticles suitable for use as a core of a SERS-activeindicator particle can be isotropic or anisotropic. As referred toherein, anisotropic nanoparticles have a length and a width. In someembodiments, the length of an anisotropic nanoparticle core is thedimension parallel to the aperture in which the nanoparticle wasproduced. In some embodiments, the anisotropic nanoparticle core has adiameter (width) of about 350 nm or less. In other embodiments, theanisotropic nanoparticle core has a diameter (width) of about 250 nm orless and in some embodiments, a diameter (width) of about 100 nm orless. In some embodiments, the width of the anisotropic nanoparticlecore is between about 15 nm to about 300 nm. Further, in someembodiments, the anisotropic nanoparticle core has a length, wherein thelength is between about 10 nm and 350 nm.

Much of the SERS literature (both experimental and theoretical) suggeststhat anisotropic particles (rods, triangles, prisms) can provide anincreased enhancement of the Raman signal as compared to spheres. Forexample, the so-called “antenna effect” predicts that Raman enhancementis expected to be larger at areas of higher curvature. Many reports ofanisotropic particles have been recently described, including silver(Ag) prisms and “branched” gold (Au) particles.

Anisotropic Au and Ag nanorods can be produced by electrodeposition intopreformed alumina templates, in a manner similar to the production ofNanobarcodes® particles (Oxonica Inc., Mountain View, Calif.). See,e.g., Nicewarner-Pena, S. R., et al., “Submicrometer metallic barcodes,”Science, 294, 137-141 (2001); Walton, I. D., et al., “Particles formultiplexed analysis in solution: detection and identification ofstriped metallic particles using optical microscopy,” Anal. Chem. 74,2240-2247 (2002). These particles can be prepared by the deposition ofalternating layers of materials, typically Au and Ag, into preformedalumina templates, and can have a diameter of about 250 nm and a lengthof about 6 microns.

SERS-active indicator particles also suitable for use in the presentlydisclosed methods include composite nanostructures, e.g., satellitestructures and core-shell structures, as disclosed in PCT InternationalPatent Application No. PCT/US2008/057700 to Weidemaier et al., filedMar. 20, 2008, which is incorporated herein by reference in itsentirety.

An advantage of the embodiments of SERS assays and devices for detectingmicroorganisms in culture samples is the variety of SERS-activenanoparticles that can be prepared, each having a unique SERS signature.Representative SERS-active indicator particles useful for the presentlydisclosed methods include, but are not limited to, SERS-active indicatorparticles from Oxonica Inc. (Mountain View, Calif.). Such SERS-activeindicator particles include a nanoparticle core labeled with SERSreporter molecules and encapsulated in a glass shell.

Representative, non-limiting reporter molecules include 4,4′-dipyridyl(DIPY), D8-4,4′-dipyridyl (d8DIPY), trans-1,2-bis(4-pyridyl)-ethylene(BPE), and 2-quinolinethiol (QSH), each of which have been disclosed asuseful Raman-active reporter dyes in U.S. Patent Publication No.2006/0038979 to Natan et al., published Feb. 23, 2006, which is hereinincorporated by reference in its entirety. Additional non-limitingexamples of suitable reporter molecules for the presently disclosedmethods include 1,2-dil(4-pyridyl)acetylene (BPA), 4-azobis(pyridine)(4-AZP), GM19, 1-(4-pyridyl)-1-cyano-2-(2-fluoro-4-pyridyl)-ethylene(CNFBPE), 1-cyano-1-(4-quinolinyl)-2-(4-pyridyl)-ethylene (CQPE), dye10, and 4-(4-hydroxyphenylazo)pyridine (136-7). A representative SERSspectrum of SERS-active nanoparticles labeled with 4,4′-dipyridyl (DIPY)is provided in FIG. 22. As shown in FIG. 22, the DIPY dye molecule has adominant peak at about 1601 cm-1.

SERS-active indicator particles suitable for use with the presentlydisclosed methods include, but are not limited to, nanoparticle corescomprising a surface enhanced Raman scattering (SERS)-active reportermolecule disclosed in U.S. patent application Ser. No. 12/134,594 toThomas et al., filed Jun. 6, 2008, and PCT International PatentApplication No. PCT/US2008/066023 to Thomas et al., filed Jun. 6, 2008,each of which is incorporated by reference in its entirety, and thevariety of SERS-active indicator particles disclosed in PCTInternational Patent Application No. PCT/US2008/057700 to Weidemaier etal., filed Mar. 20, 2008, which is incorporated herein by reference inits entirety.

In some embodiments, the SERS-active indicator particle comprises anencapsulant. SERS-active nanoparticles have a tendency to aggregate inaqueous solution and once aggregated are difficult to re-disperse.Further, the chemical composition of some Raman-active molecules isincompatible with chemistries used to attach other molecules, such asproteins, to metal nanoparticles. These characteristics can limit thechoice of Raman-active molecule, attachment chemistries, and othermolecules to be attached to the metal nanoparticle. Accordingly, in someembodiments, the presently disclosed methods comprise SERS-activeindicator particles in which the reporter molecule when affixed, e.g.,either adsorbed or covalently attached to a nanoparticle core, can becoated or encapsulated, for example, in a shell, of a differentmaterial, including a dielectric material, such as a polymer, glass,metal, metal oxides, such as TiO₂ and SnO₂, metal sulfides or a ceramicmaterial. Methods for preparing such SERS-active indicator particles aredescribed in U.S. Pat. No. 6,514,767 to Natan, which is incorporatedherein by reference in its entirety.

The thickness of the encapsulant can be varied depending on the physicalproperties required of the SERS-active indicator particle. Depending onthe particular combination of nanoparticle core, encapsulant, and dye,thick coatings of encapsulant, e.g., coatings on the order of one micronor more, could potentially attenuate the Raman signal. Further, a thincoating might lead to interference in the Raman spectrum of theassociated microorganism by the molecules on the encapsulant surface. Atthe same time, physical properties, such as the sedimentationcoefficient can be affected by the thickness of the encapsulant. Ingeneral, the thicker the encapsulant, the more effective thesequestration of the SERS-active dyes on the metal nanoparticle corefrom the surrounding solvent.

In embodiments wherein the encapsulant is glass, the thickness of theglass typically can range from about 1 nm to about 70 nm. In exemplary,non-limiting embodiments, the SERS-active indicator particles comprisegold nanoparticles having a diameter ranging from about 50 nm to about100 nm encapsulated in a sphere of glass having a thickness ranging fromabout 5 nm to about 65 nm, in some embodiments, from about 10 nm toabout 50 nm; in some embodiments, from about 15 nm to about 40 nm; and,in some embodiments, about 35 nm. The optimization of the dimensions ofthe presently disclosed SERS-active indicator particles can beaccomplished by one of ordinary skill in the art.

Further, SERS-active indicator particles comprising SERS-active dyes canbe functionalized with a molecule, such as a specific binding member ofa binding pair, which can bind to a target microorganism. Upon bindingthe target microorganism, the SERS signal of the SERS-active reportermolecule changes in such a way that the presence or amount of the targetmicroorganism can be determined. The use of a functionalized SERS-activeindicator particle has several advantages over non-functionalizedindicator particle. First, the functional group provides a degree ofspecificity to the indicator particle by providing a specificinteraction with a target microorganism. Second, the targetmicroorganism does not have to be Raman active itself; its presence canbe determined by observing changes in the SERS signal of theRaman-active dye attached to the nanoparticle core. Such measurementsare referred to herein as “indirect detection,” in which the presence orabsence of a target microorganism in a culture sample is determined bydetecting a SERS signal that does not directly emanate from themicroorganism of interest.

In other embodiments, the SERS-active indicator particle comprises aSERS-active nanoparticle as a core, with no reporter molecule orencapsulant present. The surface of the core can be functionalized witha molecule, such as a specific binding member of a binding pair, whichcan bind to a target microorganism. Upon binding the targetmicroorganism, the SERS spectrum of the target microorganism itself isdetected to confirm the presence or amount of the target microorganism.Such measurements are referred to herein as “direct detection,” in whichthe presence or absence of a target microorganism in a blood culturesample is determined by detecting a SERS signal that emanates directlyfrom the microorganism of interest.

The SERS-active indicator particles can be functionalized to bind to atarget analyte in at least two different ways. In some embodiments, theSERS-active reporter molecule, i.e., the SERS-active dye, can beconjugated with a specific binding member of a binding pair, whereas inother embodiments, a specific binding member of a binding pair can beattached directly to the nanoparticle core. In embodiments in which thenanoparticle core is at least partially surrounded by an encapsulatingshell, the binding member can be attached to an outer surface of theencapsulating shell.

C. Specific Binding Members

As used herein, the term “specific binding member,” and grammaticalderivations thereof, refers to a molecule for which there exists atleast one separate, complementary binding molecule. A specific bindingmember is a molecule that binds, attaches, or otherwise associates witha specific molecule, e.g., a microorganism of interest. When a specificbinding member of a particular type binds a particular type of molecule,the specific binding members are referred to as a “specific bindingpair.” For example, an antibody will specifically bind an antigen.Accordingly, “specific binding pair” refers to two different molecules,where one of the molecules through chemical or physical meansspecifically binds the second molecule. In this sense, a microorganismunder test is a reciprocal member of a specific binding pair.Representative binding members suitable for use with particularmicroorganisms under test are provided herein below.

Further, specific binding pairs can include members that are analogs ofthe original specific binding partners, for example, an analyte-analoghaving a similar structure to the analyte. By “similar” it is intendedthat, for example, an analyte-analog has an amino acid sequence that hasat least about 60% or 65% sequence identity, about 70% or 75% sequenceidentity, about 80% or 85% sequence identity, about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater amino acid sequence identitycompared to an analyte amino acid sequence using alignment programs andstandard parameters well known in the art. An analog of an analyte alsocan have the same function as an analyte.

A specific binding member, when conjugated, for example, with aSERS-active indicator particle, interacts with a specific microorganismunder test in a manner capable of producing a detectable Raman signaldifferentiable from when a particular microorganism is present orabsent, or when a particular microorganism is present in varyingconcentrations over time.

The term “producing a detectable signal” refers to the ability torecognize the presence of a reported group or a change in a property ofa reporter group, e.g., SERS-active reporter molecule, in a manner thatenables the detection of the binding member-microorganism complex.Further, the producing of a detectable signal can be reversible ornon-reversible. The signal-producing event includes continuous,programmed, and episodic means, including one-time or reusableapplications. The reversible signal-producing event can be instantaneousor can be time-dependent, so long as a correlation with the presence orconcentration of the analyte is established.

The binding, attachment, or association between the specific bindingmember and, for example, a microorganism, can be chemical or physical.The term “affinity” refers to the strength of the attraction between onebinding member to another member of a binding pair at a particularbinding site. The term “specificity” and derivations thereof, refer tothe likelihood that a binding member will preferentially bind to theother intended member of a binding pair (the target as opposed to theother components in the sample). Such binding between one bindingmember, e.g., a binding protein, to another binding member of a bindingpair, e.g., a ligand or analyte, can be reversible.

Further, as disclosed in U.S. patent application Ser. No. 12/134,594 toThomas et al., filed Jun. 6, 2008, and PCT International PatentApplication No. PCT/US2008/066023 to Thomas et al., filed Jun. 6, 2008,each of which is incorporated by reference in its entirety, in someembodiments, a polyethylene glycol (PEG) linker can be used to attach aspecific binding member to a SERS-active indicator particle, a magneticcapture particle, or to a solid support. In the presently disclosedmethods, a linker molecule, e.g., PEG, also can be used to attach aspecific binding member to a SERS-active indicator particle, or amagnetic capture particle. The use of a PEG linker can reducenon-specific binding in the presently disclosed assays. Eliminatingnon-specific adsorption can be a significant challenge to assayperformance. For example, in magnetic capture assays, non-specificbinding can include the process in which proteins or other biomoleculesfrom solution adhere to the surfaces of the magnetic capture particle orSERS-active indicator particle presenting binding members for the targetanalyte or the process by which the surfaces of the magnetic captureparticle and SERS-active nanoparticle adhere to one another vianon-specific interactions. In some embodiments, the PEG linker comprisesa bifunctional PEG molecule having a functional group on either terminalend of the linear molecule, separated by two or more ethylene glycolsubunits. In some embodiments, the PEG molecule comprises between 2 andabout 1000 ethylene glycol subunits. In particular embodiments, the PEGlinker comprises at least 12 ethylene glycol subunits. Further, the PEGlinker can be characterized by having a molecular weight of about 200 Dato about 100,000 Da.

Depending on the binding member, one of ordinary skill in the art wouldrecognize upon review of the presently disclosed subject matter thatlinkers other than PEG can be used. For example, alkanethiols can beused as linkers for antibodies and peptides. Short chain alkanethiols,including, but not limited to, N-succinimidyl-5-acetylthioacetate (SATA)and N-succinimidyl-5-acetylthiopropionate (SATP) can be used as linkersafter sulfhydryl deprotection. Other properties also can determine thechoice of linker, such as the length of the linker chain. For example,PEG can be desirable in that it also acts to protect the surface of thereagent and is flexible, which can enhance the ability of the reagent tobind to the analyte of interest.

In some embodiments, the specific binding member is an immunoglobulin,also referred to herein as an antibody, which comprises an antigenbinding region that binds to antigens on the target microorganism orsecreted thereby.

Antibodies and fragments thereof suitable for use in the presentlydisclosed methods and devices may be naturally occurring orrecombinantly derived and can include, but are not limited topolyclonal, monoclonal, multispecific, human, humanized, primatized, orchimeric antibodies, single-chain antibodies, epitope-binding fragments,e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv),disulfide-linked Fvs (sdFv), fragments comprising either a variablelight (VL) or variable heavy (VH) domain, fragments produced by a Fabexpression library, and anti-idiotypic (anti-Id) antibodies. In allcases, the antibody or fragment thereof will have one or morecomplementarity determining regions (CDRs) specific for the targetantigen. For purposes of the invention, a “complementarity determiningregion of an antibody” is that portion of an antibody which binds to anepitope, including any framework regions necessary for such binding, andwhich can be comprised of a subset of amino acid residues encoded by thehuman heavy chain V, D and J regions, the human light chain V and Jregions, and/or combinations thereof.

Those skilled in the art are enabled to make any such antibodyderivatives using standard art-recognized techniques. For example, Joneset al. (1986) Nature 321: 522-525 discloses replacing the CDRs of ahuman antibody with those from a mouse antibody. Marx (1985) Science229: 455-456 discusses chimeric antibodies having mouse variable regionsand human constant regions. Rodwell (1989) Nature 342: 99-100 discusseslower molecular weight recognition elements derived from antibody CDRinformation. Clackson (1991) Br. J. Rheumatol. 3052: 36-39 discussesgenetically engineered monoclonal antibodies, including Fv fragmentderivatives, single chain antibodies, fusion proteins chimericantibodies and humanized rodent antibodies. Reichman et al. (1988)Nature 332: 323-327 discloses a human antibody on which rathypervariable regions have been grafted. Verhoeyen et al. (1988) Science239: 1534-1536 teaches grafting of a mouse antigen binding site onto ahuman antibody.

D. Magnetic Capture Particles

Magnetic capture particles suitable for use with the presently disclosedembodiments can comprise from about 15% to about 100% magnetic materialsuch as, for example, magnetite, including about 15% magnetite, about20% magnetite, about 25% magnetite, about 30% magnetite, about 35%magnetite, about 40% magnetite, about 45% magnetite, about 50%magnetite, about 55% magnetite, about 60% magnetite, about 65%magnetite, about 70% magnetite, about 75% magnetite, about 80%magnetite, about 85% magnetite, about 90% magnetite, about 95%magnetite, and any integer between about 15% and about 100%. Further,the magnetic capture particles can have a diameter ranging from about100 nm to about 12 microns. In some embodiments, the magnetic captureparticles have a diameter ranging from about 400 nm to about 8 microns.In other embodiments, the magnetic capture particles have a diameterranging from about 800 nm to about 4 microns. In yet other embodiments,the magnetic capture particles have a diameter ranging from about 1.6microns to about 3.5 microns, including but not limited to, about 1.6,about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9,about 3.0, about 3.1, about 3.2, and about 3.3, about 3.4, about 3.5,and about 4.5 microns. Representative particles suitable for use asmagnetic capture particles can be obtained from Bangs Laboratories, Inc.(Fishers, Ind.), Life Technologies (Carlsbad, Calif.), or PolyscienceLaboratories (Warrington, Pa.).

Magnetic capture of the particles can be accomplished using any methodknown in the art, including, but not limited to, placing a strong magnetor inducing a magnetic field at a localized area of the assay vessel.The localized magnetic field can be induced, for example, by one or morepermanent magnets, electromagnets, and/or materials (e.g., ferrousmetals) to conduct, constrain, or focus a magnetic field. As depicted inFIG. 18, which represents one embodiment, the magnet 15 is used tolocalize the magnetic capture particle-microorganism-SERS-activeindicator particle complexes within the measurement zone 9. Incidentradiation of a desired wavelength, e.g., a laser beam, can then befocused on the pellet of concentrated magnetic captureparticle-microorganism-SERS-active indicator particle complexes and theSERS signal is obtained from the complexes.

E. Real Time System for Monitoring Growth in a Microbiological CultureSample

FIG. 24 depicts an embodiment of a real-time system 150 which providesreal-time monitoring of microorganism growth in microbiological culturesamples for the automated detection of pathogens in clinical andindustrial samples. The system 150 includes a carousel 152 that holds aplurality of culture vessels 154 within a temperature controlledenclosure. For example, up to 25 microbiological culture vessels may beused. In this embodiment, the vessels 154 are placed around theperiphery of a carousel 152, which rotates to present each vessel to apelleting and read station 156 in sequence at a programmable frequency(e.g., one reading every 10-60 minutes). The pelleting and read station156 includes a magnet assembly to form a pellet along with an opticalread head containing appropriate filters and lenses for epi-illuminationand signal collection. For example, illumination may be provided by a785 nm wavelength-stabilized laser, and collected signal is detected ona spectrometer appropriate to Raman spectroscopy. After pelleting andreading a given sample, the carousel 152 rotates to present the nextsample in the carousel to the pelleting and read station 156. Thesequence continues until all samples in the carousel 152 are read, atwhich point the carousel enters a spinning mode wherein the carouselrotates continuously until the next measurement cycle. The carousel andpelleting and read station are mounted to an arm which extends from arocking platform. The resulting “offset rocking” motion providesagitation through both linear and rocking motion. The rocking platformoperates at a selected frequency for all phases of a measurements cycle,for the duration of the experiment. The agitation serves multiplepurposes. First, from the end of one measurement cycle to the start ofthe next, it ensures mixing of the SERS and magnetic particles with thesample and culture media, allowing the formation of bindingmember-microorganism-indicator particle complexes. Second, after thepellet has been read, it enables the dispersion of the magneticparticles back into solution. Third, during pelleting, agitation carriesmagnetic particles in the fluid from various spatial points within thesample vessel into the region of the localized magnetic field, ensuringthat magnetic particles are collected from regions of the sample outsideof the localized magnetic field. Finally, in samples containingparticulates (e.g. resins, charcoal, or calcium carbonate), agitationprior to and during pelleting can prevent these particulates fromsettling into the detection region and interfering with the opticalsignal. As the target organism concentration increases throughout theenrichment process, detection and identification of the microorganism byoptics, such as SERS technology, occurs as soon as the microorganismconcentration reaches the detection threshold of the technology. Theability to continuously monitor the SERS signal during culture ensuresthat the minimal required culture time is used and that the instrumentcan automatically alert the user when a pathogen or microorganism isdetected and identified.

FIG. 25 shows another embodiment of a system 200 configured to process aplurality of samples. In this particular embodiment, each of themicrobiological culture samples is processed in parallel and incubatedwithin the same thermal zone. In this embodiment, the sample tubes arealigned in the same horizontal plane. The system 200 may be placedwithin an enclosure that forms the thermal zone.

All samples agitate together for the reagent binding, pelleting, andpellet dispersal phases. In one embodiment, after pelleting, agitationstops for all samples, and they are read in succession. The parallelprocessing requires a different sample arrangement than the carouselused in the first system 150 configuration. Here, the sample tubes 202are positioned adjacent to each other in a flat tray 204. In thisembodiment, agitation is by linear reciprocation along the longitudinalaxis of the tubes 202, which may be programmed for different frequenciesand profiles throughout the assay. This allows different types andlevels agitation for the pellet formation, pellet dispersal, and reagentbinding phases. It also permits the agitation to be stopped for reading.The programming of different agitation at each phase is made possible bythe parallel sample processing approach.

The system 200 shown in FIG. 25 includes a magnet assembly 206 that isconfigured to pivot into a position adjacent to the tubes 202 forpelleting and then pivot away from the sample tubes for interrogation.For the system 200 embodiment shown in FIG. 25, the opticalinterrogation can occur with the magnet assembly 206 pivoted away fromthe sample tubes 202 after pellet formation to allow access of theoptical read head 208 to the measurement zone of the sample tubes.Withdrawing the magnet assembly 206 is possible in this configurationbecause the samples are not agitating during reading. Alternately, apair of magnets may be arranged in such a way as to provide a slotthrough which the readings are taken by the read head 208, with themagnets maintained in position after pelleting. Thus, the read head 208is configured to move along the slot between the magnets to interrogateeach tube. This offers advantages by removing the need for moving themagnets between pelleting and reading of the tubes.

FIGS. 26-29 illustrate another embodiment of a system 250 for automatedand real-time monitoring of microorganism growth in culture samples. Thesystem 250 is configured to automatically process one or more differentassays simultaneously. In general, the system 250 includes a pluralityof incubators 252 serviced by a single pelleting/read assembly 254 thatmoves behind the incubators to service (pellet and read) each tray 256one at a time in a detection zone. Each incubator 252 is configured toreceive a tray 256 holding a plurality of sample tubes 258. Each tray256 of tubes is configured to move from the incubator 252 to thepelleting/read assembly 254 where pellets are formed and data collectedin the detection zone. The pelleting/read assembly 254 comprises amagnet assembly 260 to form pellets and the optical components (e.g.,Raman optics, laser, and spectrometer) to collect the optical signals.

In one embodiment, the system 250 includes a plurality of incubators252. Various assays may incubate culture samples at differenttemperatures. Thus, the system 250 may include a plurality of thermalzones 262 (incubation zones) that can operate at different temperatures,wherein each zone includes one or more incubators. The assays in eachincubator 252 are processed simultaneously, wherein each incubator mayinclude one or more trays 256 holding one or more sample tubes 258.However, the sample tubes 258 may not necessarily be processed in abatch. In this regard, each sample tube 258 can be placed in theincubator 252 at a different time, thereby having a different startingtime for its test period. The sample tubes 258 may all be exposed to thesame repeating test cycle during their test periods. The majority ofsample tubes 258 may be introduced together in batches. As shown inFIGS. 26 and 27, the system includes four incubators 252 that aredivided into two thermal zones 262. The thermal zones 262 may bearranged vertically such that two incubators 252 are associated with arespective thermal zone. Identical thermal zones 262 are stackedvertically as shown in FIG. 27. However, it is understood that thethermal zones 262 may be arranged horizontally, or side-by-side, ifdesired. Each zone 262 may include one or more incubators 252 maintainedat identical incubator levels that operate at a common temperature. Inone exemplary embodiment, the zones 262 are configured for processingSalmonella and Listeria assays, which are maintained at differenttemperatures (e.g., about 42° C. and 30° C., respectively). Thus, thesystem 250 is configured to process different sample tubes 258 (e.g.,detection vials) regardless of the type of assay.

In one embodiment, each incubator 252 forms an enclosure suitable forreceiving a tray 256 therein and maintaining a predetermined temperaturenecessary for culturing a particular sample. One example of an incubator252 is shown in FIG. 34. The incubator 252 may include a base block 264,including the bottom and side surfaces, front and rear doors 266, 268,and a top surface 270. The incubator 252 may be formed of a variety ofmaterials suitable for providing a temperature controlled enclosure. Forexample, the incubators 252 may be constructed from a single machinedmetal base block 264 (e.g., aluminum) that forms the bottom and sides ofthe temperature controlled region, while a metal plate (e.g., aluminum)forms the top surface 270. The incubator 252 may also include a channel272 on the left side of the base block that is configured to support abelt drive 274 for oscillating the tray 256 in a Y-direction undercontrol of a Y-stage 278. The range of motion of the tray 256 extendsbeyond the heated zone depth to provide tray stroke into the pellet/readarea behind the incubator 252. Thus, the range of motion of each tray256 will be based on the amount of travel needed to properly agitate thetubes 258 as well as reposition the tubes outside of the incubator 252for pelleting and image analysis by the pelleting/read assembly 254.

In one embodiment, the incubator 252 includes a front door 266 and arear door 268, wherein the doors cooperate with the top surface 270 andthe base block 264 to form an enclosure. The front door 266 isconfigured to be selectively opened and closed by an operator (see e.g.,FIG. 31). For example, the front door 266 may be configured to swingdown so that the tray 256 can extend from the front of the incubator 252a minimum distance and the door will not obstruct tube access orvisibility. The front door 266 may be mounted using a variety oftechniques to facilitate opening and closing. For instance, the frontdoor 266 may be mounted on torsion spring loaded hinges that close thedoor upon tray withdrawal into the incubator 252. A pusher mechanism mayextend from one or both sides of the front of the tray to aid in openingthe door. According to one aspect, a flag is located inside of the frontdoor 266 that is configured to interrupt an optical sensor mounted onthe interior side wall of the incubator 252 to sense when the front dooris closed.

Similarly, the rear door 268 may be configured to open and close uponthe tray 256 exiting and reentering the rear of the incubator 252 (seeFIG. 40). Thus, as the tray 256 exits the incubator 252, the tray isconfigured to push the rear door 268 at least partially open. Like thefront door 266, the rear door 268 may be mounted on torsion springloaded hinges, and a feature on the rear of the tray 256 may beconfigured to push the rear door open as the tray emerges from the rearof the incubator 252. A sheath 276 is configured to receive the tray 256upon exiting the incubator 252, and is configured for motion along aZ-axis as explained in further detail below. Once the rear door 268 ispartially open, the sheath 276 may be configured to fully open the reardoor as the sheath rises along the Z-axis to align with the incubator252. With the sheath 276 holding the door open, the tray 256 is free tomove in the pellet/read area without interference from the rear door268. The rear door 268 is configured to close when the tray 256 isretracted into the incubator 252 and the sheath 276 lowers. Theincubator 252 may include a sensor for indicating that the tray 256 isproperly positioned therein. For instance, a flag on the inside of therear door 268 may be configured to interrupt an optical sensor mountedon the interior side wall of the incubator to sense when the rear dooris closed. This sensor may also be used as a home indicator for the tray256. Thus, the rear door sensor may establish the tray 256 position asin or out of the rear of the incubator 252. A second home sensor maylocate home for each tray 256 relative to the pelleting and opticalhardware during each excursion into the pelleting/read region.

Because each incubator 252 is temperature controlled, the incubator maybe wrapped by an insulating material (e.g., a closed cell foaminsulation). Each of the thermal zones 262 may also be separated by aninsulating material, which is useful when the zones are maintained atdifferent temperatures. There may also be gaps between zones to limitcross talk between zones. In addition, the insulating material may beused to prevent thermal interaction when one incubator door 266 or 268is open to the front or rear and the other is closed. Insulating spacersmay separate incubators in a zone 262. Similarly, zones 262 may also beseparated using spacers and insulating material.

Each incubator 252 is heated using a heating element. For example, theheating element may be configured to conduct heat through the base block264 or provide heated air within the incubator. According to oneembodiment, the heating element is a flat heating element adhered to orotherwise integrated with the bottom surface of the base block 264. Thepower distribution of the heating element may be tailored to minimizethermal gradients across the tubes 258 in the tray 256 to compensate forthermal loss through the Y drive 278 components on the left side of theincubator 252. Each incubator 252 may be provided with one or moresensors for monitoring temperature therein, such as the temperature ofthe base block 264 and/or the air within the incubator.

As discussed above, each incubator 252 is configured to receive arespective tray 256 therein. FIGS. 30-32 illustrate exemplaryembodiments of trays 256 suitable for positioning within a respectiveincubator. A tray 256 in each incubator 252 is configured to accommodatea plurality of tubes 258. In the illustrated embodiment, the tray 256holds 15 tubes 258 such that each thermal zone 262 holds 30 tubes,although any number of tubes may be used. The tubes 258 may be arrangedhorizontally in a planar array in each of the trays 256. In oneembodiment shown in FIG. 31, a technician manually places sample tubes258 into removable sample trays 256. The technician then places thetrays 256 into the incubators 252. An assay is complete when a positiveresult is determined or when the result remains negative for apredetermined period of time. When the assay is completed, thetechnician removes the trays 256 to be refilled with new tubes 258.Alternatively, the technician may remove and/or add tubes to the trayindividually as needed without stopping assays already in progresswithin other tubes in the tray or adjacent incubators. Positive samplesmay be segregated for further analysis. In another embodiment, the trays256 are not removable. Thus, the tubes 258 can be placed individuallyinto a non-removable tray 256 in each incubator 252. In yet anotherembodiment, the tray 256 may be unnecessary where the incubator 252includes suitable means for holding the tubes 258 therein.

The arrangement of the tubes 258 horizontally and side-by-side in thetrays 256 facilitates loading individual sample tubes or trays from thefront of the incubator 252. Front loading avoids using bench space orisle space in front of the system, as a top-loaded tray would need toextend out the front of the system nearly the length of a tube tofacilitate top loading. Further, the tray and sliding support would needto withstand high loads when a user exerts excessive pressure on thecantilevered extended tray.

Each tray 256 may be a variety of sizes and configurations for holdingtubes 258 and facilitating placement within the incubator 252. Forinstance, FIG. 31 illustrates that the tubes 258 are inserted withinrespective slots 284 defined in the tray 256. The tubes 258 may be heldin place using a force or an interference fit or biasing elementsdisposed within the tray 256. Where the trays 256 are removable from theincubator 252, the trays may include one or more gripping features 286that allow a technician to hold the trays and manipulate the trayswithin the incubator, as shown in FIGS. 33A-33C.

In one embodiment, the tray 256 includes longitudinal slots 284 suchthat a portion of the tubes 258 is visible through the tray.Longitudinal slots 284 also allow the tubes 258 to protrude below thebottom surface of the tray 256 to provide a contact area with thepelleting magnets 288. To ensure sufficient contact with the magnetsacross the tray 256, each tube 258 may have vertical compliance in thetray. For example, a spring may hold the tube 258 down against themagnets as they rise to meet the tube. The spring may also retain thetubes 258 in the tray 256 by friction against the oscillatory traymotion.

According to one embodiment, the trays 256 are configured to oscillatehorizontally along a Y axis in each incubator 252 under the control of aY-stage 278 to agitate tubes containing a sample, culture medium, andreagent. This horizontal motion may fulfill several functions:

-   -   a) Agitation for kinetic mixing in the incubator 252;    -   b) Extending the tubes out the front of the incubator 252 for        operator loading and unloading;    -   c) Extending the tubes out the rear of the incubators 252 to the        pelleting/read assembly 254;    -   d) Agitating the tubes 258 to disperse settled materials—e.g.,        solid components of media or samples;    -   e) Agitating the tubes 258 and magnets 288 in the pellet/read        assembly 254 to form pellets;    -   f) Positioning the tubes 258 over the read head 290 for data        collection;    -   g) Positioning tube labels over a bar code reader for sample ID;    -   h) Positioning the pellets over a camera to visualize pellets        for internal controls, image-based detection methods, or remote        diagnostics;    -   i) Agitating the tubes 258 to disperse pellets after reading;    -   j) Operating the incubator front door 266;    -   k) Operating the incubator rear door 268;    -   l) Circulating air in the incubator 252 to reduce temperature        gradients.

As shown in FIG. 34, the system includes a Y-stage 278 for moving thetrays 256 along a Y-axis, including for oscillating or agitating thetrays and moving the trays in and out of the rear of the incubator 252.As shown in FIG. 27, each incubator 252 may include a respective Y-stage278 disposed along the Z-axis so as to be spaced vertically from oneanother. The tray 256 may be coupled to the Y-stage 278 using one ormore carriages 282. For example, the tray 256 may be cantilevered from acarriage 282 mounted on a linear rail 292, wherein the linear rail ismounted to the incubator base block 264. The Y-stage 278 includes amotor 280 for driving a belt 274 around timing pulleys at both ends ofthe rail 292. It is understood that the Y-stage 278 is configured toagitate that trays 256 at a variety of frequencies and amplitudesdepending on the particular assay and application. For example, thetrays 256 may be oscillated more slowly while in the incubators 252 thanwhen positioned in the pelleting/read assembly 254.

The Y-stage 278 components may also be enclosed by an insulatingmaterial, while the motor 280 driving the Y-stage is outside theinsulated area. The trays 256 and carriage 282 may move through anopening in the insulating material.

The system 250 also includes a Z-stage 294 located behind the incubators252 (see FIGS. 39, 42, and 43). The Z-stage 294 is configured to carrythe sheath 276, magnet assembly 260, optical components (e.g.,spectrometer 308, Raman probe 310, etc.), and X-stage 296 along aZ-axis. The Z-stage 294 may include a carriage 298 configured to ride ona vertical linear rail 300. A motor 302 is configured to raise and lowerthe Z-stage in a Z direction (e.g., using a linear screw and nutdisposed within shaft 304 coupled to a Z-stage bracket 306). A sensormay be used to indicate when the Z-stage 294 is at the bottom of itstravel in the Z-direction. The spectrometer 308, magnet assembly 260,sheath 276, and X-stage 296 are mounted to the Z-stage bracket 306 andall travel together in the Z-direction as a unit. The Z-stage 294 isconfigured to move in the Z-direction to accommodate each tray 256 thatexits the incubator 252. The Z-stage 294 is also configured to travelbelow the bottom incubator 252 sufficiently to permit the bottomincubator rear door 268 to close.

The system 250 also includes a magnet assembly 260, as shown in FIGS.42, 45, and 46. The magnet assembly 260 may include one or more magnets288 mounted to a magnet frame 318 configured to apply a magnetic fieldto the tubes 258, thereby facilitating the formation of a pellet withineach tube. For example, FIGS. 45 and 46 illustrate a pair oflongitudinal magnets 288 spaced apart a sufficient distance to permitthe read head 290 to extend therethrough to obtain a reading. Thus, themagnets 288 may remain in position following pelleting and while thetubes 258 are being read by the read head 290. Each longitudinal magnet288 may be a single magnet or a collection of a plurality of magnetsarranged end to end.

Pellets may be formed when magnets 288 are brought into contact with, orwithin close proximity to, the bottom of the horizontally oriented tubes258. The tubes 258 and magnets 288 gently oscillate during pelletformation to ensure the magnetic particles in suspension pass throughthe magnetic field and are attracted to a magnetic field focal point.According to one embodiment, the magnet assembly 260 is mounted to acarriage 312 that rides in the Y-direction on a rail 314 affixed to theZ-stage bracket 306 (see FIG. 46). The rail 314 may be parallel to theY-stage rail 292.

When the tray 256 extends out of the rear of the incubator 252 and intothe pelleting/read assembly 254, the Z-stage 294 may be raised frombelow along a Z-axis. As shown in FIGS. 42 and 47, a pin 316 extendingoutwardly from the magnet frame 318 is configured to engage with a holein the underside of the tray 256. Once engaged, the magnet frame 318 iscoupled with the tray 256, and the magnet frame and tray are able tomove together in the Y-direction along rail 314. Therefore, in oneembodiment, the pelleting/read assembly 254 is configured to process onetray 256 at a time. The pelleting oscillation amplitude may varydepending on a number of factors specific to a particular assay. In oneexample, the oscillating amplitude may be up to about 50 mm. The magnetframe 318 and tray 256 may be coupled at a position of full travel ofthe Y-stage in the Y-direction, while the center of the pelletingoscillations may be located forward from the coupling location toaccommodate ½ the amplitude in the Y-direction.

In one embodiment, a small amount of relative motion between theoscillating tubes 258 and the magnets 288 allows the magnetic field togather the magnetic particles into a tighter pellet. Thus, a loosecoupling between the tray 256 and magnet frame 318 may be desirable.Such a coupling may be implemented, for instance, by mounting the frame318 on a block held in a slot in the frame 318 between two springs. Asthe tray 256 oscillates fore and aft in the Y-direction, the frame 318moves in relation to the tray as the springs alternately compress in asecond oscillatory motion. The loose coupling stroke may be, forexample, about 5 mm. The spring constants will be selected to providethe optimal oscillation frequency.

The magnet assembly 260 is configured to pellet each of the tubes 258 inthe tray 256 simultaneously, according to one embodiment of the presentinvention. The magnets 288 may be configured to remain in place whilethe tubes 258 are being read by the read head 290. Alternatively, thepellets may be adequately persistent to permit the magnet 288 or tubes258 to be moved away from one another for reading.

FIG. 41 shows one example of an X-stage 296 that is coupled to theZ-stage 294. Thus, the X-stage 296 is configured to be moved in the Zdirection by the Z-stage. The X-stage 296 also facilitates motion of theread head 290 in the X-direction for reading each of the tubes 258. TheX-stage 296 scans the read head 290 under the array of tubes 258 tocollect assay data after pellets have been formed. During pelleting, theX-stage 296 is moved to an X-position that allows a coupled tray 256 tobe moved without interference with the read head 290 and the magnetassembly 260. After pelleting, with the tray 256 held stationary, theX-stage 296 translates to each tube 258, pausing to collect data untilall tubes are read. The X-stage 296 is then repositioned to its startposition before the tray 256 is moved. In one exemplary embodiment, theX-stage drive (e.g., motor 320 and belt 322) is mounted in a channel 324in the Z-stage bracket 306 and utilizes a similar design as that of theY-stage 278. The read head 290 is mounted to a carriage 326 that isconfigured to move along a rail 328 of the X-stage 296. A flexiblesleeve 330 may be used to route the read head 290 electrical cables andflexible fiber optic cable from the Z-stage 294 to the moving read head.The flexible sleeve 330 is configured to not only protect the fiberoptic bundle but also allow the fiber to flex in the X-direction andprovide a desired bend radius.

According to another embodiment, the X-stage 296 is configured to carrya bar code reader (not shown) for reading a bar code or other identifieron each of the tubes 258. For example, the bar code reader may be usedto confirm that the tube 258 is in the correct thermal zone 262, therebypreventing false negatives. The bar code could include other data, suchas identification and assay information. As discussed above, the tubes258 may include longitudinal slots 284 that facilitate such reading by abar code reader. Additionally, the bar code reader may provide imagingdata on pellets for internal controls, image-based detection methods,and/or remote diagnostics.

As discussed above, a sheath 276 is configured to receive each tray 256as the tray exits the rear of the incubator 252. In particular, theinsulated sheath 276 is carried on the Z-stage 294 and is configured toalign with each incubator 252 to surround the tray 256 when it extendsout the rear of the incubator into the pelleting/read region 254. Theinsulated sheath 276 minimizes the tray temperature change while thetray 256 is extended out of the incubator 252. The sheath 276 bothprovides an insulated sleeve and blocks air flow from cooling the tray256 and tubes 258 contained therein. Also, the sheath 276 is constructedfrom materials that minimize its thermal mass and thus the heat energyexchange with a tray 256 at a different temperature from than that ofthe preceding zone. For example, the sheath 276 may comprise a thinaluminum structure surrounding by an insulating material. In onespecific embodiment, a tray at about 30° C. entering a sheath at about42° C. will not increase in temperature by more than about 0.5° C.

There may be a gap 332 defined between the incubator 252 and the alignedsheath 276 such that the incubator cannot fully regulate the temperaturein the sheath. In those instances where the temperature in the incubator252 is higher than ambient temperature, the air surrounding the sheath276 may be cooler than the tray 256, so any thermal transfer from thesheath will be toward a lower tray temperature. However, some assayshave an acceptable temperature tolerance should there be variationsresulting from movement of the tray 256 from the incubator 252 into thesheath 276. For example, assays are more tolerant of brief negativetemperature dips, e.g., about −2° C., than temperature rises, e.g.,about +0.5° C. for Salmonella at 42° C. and Listeria at 30° C. Thus, itmay be unnecessary to form a good thermal seal with the incubator 252 aslong as negative excursions are within acceptable tolerances.

In addition to surrounding the extended tray 256, the sheath 276 mayalso enclose the magnet assembly 260 as shown in FIG. 42. Thus, thesheath 276 may be sized to enable the Z-stage 294 to move verticallywith the magnet assembly 260 for coupling and uncoupling the tray 256,as discussed above. Moreover, the sheath 276 may include cutouts toprovide clearance for the read head 290 along the bottom of the sheathand for the carriages 282 moving along the side via the Y-stage 278. Thetop of the sheath 276 may also include cutout for the rear incubatordoor 268.

The aforementioned components of the system 250 may be enclosed in acabinet 334, as shown in FIGS. 48A and 48B. A skin forms a cabinet 334around the incubator 252 and pelleting/read region 254 to control airflow and add thermal control. The cabinet 334 also may also provide asafe enclosure in which the optical components operate (e.g., laser).The system 250 may further include one or more visible or audiblesignals for indicating the status of the assays. For example, one ormore LED's 336 may be used to indicate that the assay is in progress andwhen a positive result occurs. Each tube 258 and/or tray 256 may includean associated LED 336 for such a purpose. Different LED colors may beused for different indications, wherein the colors may be visible whenthe front door 266 of the incubator 252 is open or closed.

The incubators 252 typically are maintained at a temperature that ishigher than ambient. To aid in achieving this temperature difference andensure excess heat is not delivered to trays 256 extending into thesheath 276, an air flow path may be employed. Fans with filters may alsobe used to pressurize the cabinet 334 to reduce dust infiltration, andother heat dissipation techniques may be used for components such as themotors. Other techniques, such as a thermal electric cooler may be usedto further cool the cabinet 334.

Various electrical components may be used for interfacing with andcontrolling the system 250 as known to those of ordinary skill in theart. For example, various motor driver boards may be used to control themotors and provide the interface to the other devices such as sensorsand encoders. Other boards may be used to provide additionalfunctionality such as providing power and interface signals for the readhead 290 and the spectrometer 308 as well as driving heaters and readingthe associated thermistors. Additionally, the system 250 may employvarious other components such as a microcontroller for controlling thesystem in an automated manner as known to those of ordinary skill in theart.

E. Agitation and Pelleting Techniques

FIG. 49 depicts methods of agitation and pelleting according to anembodiment of the invention which have been developed that minimize thereagent volume and obtain a reading that is representative of a largevolume (e.g., up to 250 mL). This is accomplished primarily by agitatingthe culture vessel along its longitudinal axis during the application ofthe magnetic field. The pellet is formed on the side of the tube, alongthe direction of the longitudinal axis. A combination of sample-to-tubevolume (e.g. 1:2), tube length/width aspect ratio (e.g., 7:1), andagitation parameters results may be selected to optimize performance.Agitation promotes more efficient pellet formation by ensuring thatparticles from the larger volume are brought into close proximity withthe magnet. In addition, agitation helps keep non-complexed cells,microorganisms, and loose solids (e.g. resin in blood culture samples)out of the pellet by applying a force away from the magnetic fielddirection.

FIG. 50 depicts one embodiment of a device for forming and interrogatingthe magnetic pellet. This device may be used in the carousel system 150embodiment shown in FIG. 24. In the embodiment illustrated in FIG. 50,the magnet assembly consists of a stack of ring magnets surrounding andcollinear with the portion of the optical read head containing theobjective lens. A hollow conical steel tip focuses the magnetic field atthe tip of the cone, causing the pellet to be formed at the focus of theread head. This arrangement automatically aligns the pellet with thefocus of the read head and relaxes the constraints on the alignment oftube, magnet, and read head. Consistent alignment of pellet, magnet, andread head are important for consistent measurements across all samplesover the course of the assay, and this design provides reliable andrepeatable pellet formation across sample tubes, over multiple readsduring the course of an assay, and across instruments.

FIGS. 51 and 52 show alternate methods of forming the pellet that havebeen used in accordance with the systems described above. The alignmentof the tubes in a horizontal plane allows the optical signal to be readwith the magnets either withdrawn after pelleting or maintained in thepelleting position. This allows various magnet geometries to be tested.Two have proven to be especially effective. One, termed “North-up” usesa bar magnet with magnetization directed normal to the tube (FIG. 52).This forms a symmetric circular pellet ideal for reading with the readhead. The other geometry is termed “North-facing-North pair” (FIG. 51),in which two bar magnets are positioned adjacent and parallel to eachother. The magnetization is directed along the line normal to themagnets through the thickness of each magnet, such that the North polesface each other. With proper spacing between the magnets, the region ofhighest field gradient is in the region between the magnets, resultingin a pellet focused in the region between the magnets, which is easilyaccessible for reading with the magnets in place.

In another embodiment of the invention a camera is also added to thetesting station to monitor the formation and size of the pellet duringSERS-HNW assay which contains conjugated SERS indicator particles andmagnetic beads and the targeted pathogen within a culture vessel. Thepellet size increases, and in some cases the pellet disappears, from thecamera view as the HNW assay progresses. The growth in pellet sizeand/or disappearance of the pellet is an indication of the presence ofthe targeted pathogen. Images captured during analysis of samples thatcontain conjugated SERS indicator particles and magnetic beads with nopathogen show no change in pellet size and no pellet disappearance. Thismethod of pathogen detection can be used alone or in conjunction withanother detection method such as the previously described SERS analysisas a means of validation.

Embodiments of the presently disclosed methods can be conducted with anysuitable spectrometers or Raman spectrometer systems known in the art,including, for example, a Multimode Multiple Spectrometer RamanSpectrometer (Centice, Morrisville, N.C., United States of America),such as the Raman spectrometer system disclosed in U.S. Pat. No.7,002,679 to Brady et al., which is incorporated herein by reference inits entirety. Other non-limiting examples of suitable spectrometers orRaman spectrometer systems include the Hamamatsu C9405CA and the IntevacReporteR, and include both fiber-coupled and free-space opticalconfigurations. Additional instrumentation suitable for use with thepresently disclosed SERS-active indicator particles is disclosed in PCTInternational Patent Application No. PCT/US2008/057700 to Weidemaier etal., filed Mar. 20, 2008, which is incorporated herein by reference inits entirety.

Representative methods for conducting magnetic capture liquid-based SERSassays are disclosed in PCT International Patent Application No.PCT/US2008/057700 to Weidemaier et al., filed Mar. 20, 2008, which isincorporated herein by reference in its entirety. Such methods caninclude referencing and control methods for compensating for variationsin magnetic pellet size, shape, or positioning, and methods forgenerating improved Raman reference spectra and spectral analysis inmagnetic pull-down liquid-based assays, as also disclosed inPCT/US2008/057700. Further, multiple reporter molecules can be used tocreate an internal reference signal that can be used to distinguishbackground noise from signal detection, particularly in samples thatexhibit or are expected to exhibit a relatively weak signal.

Further, as disclosed in U.S. patent application Ser. No. 12/134,594 toThomas et al., filed Jun. 6, 2008, and PCT International PatentApplication No. PCT/US2008/066023 to Thomas et al., filed Jun. 6, 2008,each of which is incorporated by reference in its entirety, dyessuitable for use as reporter molecules in SERS-active indicatorparticles typically exhibit relatively simple Raman spectra with narrowline widths. This characteristic allows for the detection of severaldifferent Raman-active species in the same sample volume. Accordingly,this feature allows multiple SERS-active indicator particles, eachincluding different dyes, to be fabricated such that the Raman spectrumof each dye can be distinguished in a mixture of different types ofindicator particles. This feature allows for the multiplex detection ofseveral different target species in a small sample volume, referred toherein as multiplex assays.

Accordingly, in some embodiments, more than one type of binding membercan be attached to the SERS-active indicator particle. For example, thetype of binding member attached to the SERS-active indicator particlecan be varied to provide multiple reagents having different affinitiesfor different target microorganisms. In this way, the assay can detectmore than one microorganism of interest or exhibit differentselectivity's or sensitivities for more than one microorganism. TheSERS-active indicator particle can be tailored for culture samples inwhich the presence of one or more microorganisms, or the concentrationsof the one or more microorganisms, can vary.

A SERS assay reagent can include more than one type of label, e.g., morethan one type of SERS-active reporter molecule, depending on therequirements of the assay. For example, SERS-active reporter moleculesexhibiting a Raman signal at different wavelengths can be used to createa unique Raman “fingerprint” for a specific microorganism of interest,thereby enhancing the specificity of the assay. Different reportermolecules can be attached to nanoparticle cores which have attachedthereto different specific binding members to provide a reagent capableof detecting more than one microorganism of interest, e.g., a pluralityof microorganisms of interest.

In an embodiment of the invention, the multiplexing capabilities of theSERS HNW technology are used to identify six of the most commonorganisms causing blood stream infections. Six different types or“flavors” of SERS-active indicator particles are present in a bloodculture bottle, each conjugated with antibodies specific to one of thesix organisms to be detected. Also in the vessel are magnetic captureparticles capable of forming sandwiches with the SERS-active indicatorparticles. The magnetic capture particles can be configured so thatthere is a common capture antibody or set of antibodies that sandwichmultiple SERS-active indicator particles or alternatively, there couldbe six separate magnetic conjugates present in the vessel, with eachmagnetic conjugate uniquely capable of forming a sandwich with each ofthe six SERS-active indicator particles. When a magnetic pellet isformed and the SERS signal from the pellet is read, the measured Ramanspectrum will be a contribution from each flavor of SERS-activeindicator particle present in the pellet; the presence of a SERS-activeindicator particle indicates the presence of the microorganism for whichthe SERS-active indicator particles is specific. Deconvolutionalgorithms can efficiently distinguish the spectra of the six individualSERS-active indicator particles from the measured aggregate spectrum.

FIG. 53 shows a schematic of a multiplexed embodiment using only twoSERS-active indicator particles. In a preferred embodiment, a standardgas sensor (e.g. BACTEC™) is retained, so that both the SERS signal andthe pH sensor signals are simultaneously monitored. This enablesefficient detection of any microorganism that is not recognized by theSERS HNW antibodies.

As further disclosed in PCT International Patent Application No.PCT/US2008/057700 to Weidemaier et al., filed Mar. 20, 2008, in thepresently disclosed assays involving SERS-active indicator particles,the SERS spectra can be amplified through the addition of a secondaliquot of reporter molecules capable of generating a detectable signaland having associated therewith at least one specific binding memberhaving an affinity for the at least one SERS-active reporter moleculeassociated with the one or more SERS-active indicator particles priorto, concurrent with, or subsequent to disposing the sample and/or the atleast one SERS-active reporter molecules therein, wherein the secondaliquot of reporter molecules is the same as the at least oneSERS-active reporter molecules associated with the SERS-active indicatorparticles. In some embodiments, the second aliquot of reporter moleculescomprises a SERS-active reporter molecule associated with a SERS-activeindicator particle capable of producing a SERS signal. In thoseembodiments wherein a second aliquot of reporter molecules is disposedinto the assay vessel, the specific binding member of the second aliquotof reporter molecules does not recognize the one or more specificbinding members comprising the capture zone or attached to the magneticcapture particles.

F. Workflow Examples

According to one exemplary embodiment, a culture sample for detectingand identifying Salmonella may be provided in conjunction with theaforementioned embodiments. In one embodiment, Salmonella is firstcultured in a non-selective media within the enrichment vessel, followedby a biocontained transfer into a detection vial containing thedetection reagents and a second, selective media. Generally, theSalmonella testing includes adding media with optional supplement into amedia preparation vessel. The media is then dispensed into theenrichment vessel and a sample is added into the enrichment vessel.Optionally, the sample is homogenized (e.g., by stomaching or blending)prior to addition to the enrichment vessel. In this case, the media fromthe media preparation vessel is added along with the sample to thehomogenizer. Following homogenization, the sample is transferred intothe enrichment vessel, and a lid is attached to the vessel. Once mediaand sample have been added to the enrichment vessel and the enrichmentvessel lid has been attached, a bar code on the vessel may be read forchain of custody identification purposes. The enrichment vessel is thenincubated for a predetermined period of time. Following incubation, theenrichment vessel and a detection vial may be scanned with a bar codereader. The detection vial includes a selective media and detectionreagents that are particular to detecting Salmonella. In the case wherethe media in the detection vial is dehydrated, reconstitution fluid isadded to the detection vial, and the vial is inverted for mixing. Theenrichment container is then tilted to fill a respective reservoir witha desired amount of sample (e.g., 100 μL). The detection vial isinserted into the enrichment vessel to engage a needle within theopening for a biocontained transfer of the sample into the detectionvial. The detection vial is then inserted within a real-time automatedsystem for incubation and automated testing of the sample, includingpelleting and optical analysis of the sample. Upon detection of apositive sample, the detection vial may be removed, scanned by a barcode scanner, and routed for further processing.

In an alternate exemplary embodiment, a culture sample for detecting andidentifying Listeria may be provided in conjunction with theaforementioned embodiments. In a preferred embodiment, culture ofListeria within the detection vial occurs in the same media that is usedin the enrichment vessel, so that a single media is used throughout theworkflow. Generally, the Listeria testing includes adding media withoptional supplement into a media preparation vessel. The media is thendispensed into the enrichment vessel and a sample is added into theenrichment vessel. Optionally, the sample is homogenized (e.g., bystomaching or blending) prior to addition to the enrichment vessel. Inthis case, the media from the media preparation vessel is added alongwith the sample to the homogenizer. Following homogenization, the sampleis transferred into the enrichment vessel, and a lid is attached to thevessel. Once media and sample have been added to the enrichment vesseland the enrichment vessel lid has been attached, a bar code on thevessel may also be read for chain of custody identification purposes.The enrichment vessel is then incubated for a predetermined period oftime. Following incubation, the enrichment vessel and a detection vialmay be scanned with a bar code reader. The detection vial includesdetection reagents that are particular to detecting Listeria. Theenrichment container is then tilted to fill a respective reservoir witha desired amount of sample (e.g., 5 mL). The detection vial is insertedinto the enrichment vessel to engage a needle within the port for abiocontained transfer of the sample into the detection vial. Thedetection vial is then inserted within a real-time automated system forincubation and automated testing of the sample, including pelleting andoptical analysis of the sample. Upon detection of a positive sample, thedetection vial may be removed, scanned by a bar code scanner, and routedfor further processing.

G. Reconstitution Station

FIGS. 93-95 illustrate reconstitution stations that may be used in aconjunction with embodiments of the present invention. As discussedabove, reconstitution fluid may be added to the detection vial where themedia in the detection vial is dehydrated. The reconstitution stationsfacilitate metering of a desired volume and enable the addition of thereconstitution fluid without exhausting a vacuum retained within thedetection vial. FIG. 93 illustrates an embodiment where thereconstitution station 350 includes a bladder 352 or similar secondaryreservoir with a pinch valve 354. The reconstitution station 350 isgravity fed such that fluid is configured to travel from the mainreservoir, through tubing 358, and into the bladder 352 when the valve354 is open. For example, a lever 356 may be rotated clockwise to engageor pinch the tubing 358 to close off the tubing at the valve 354. A useris then able to insert the detection vial 360 into the access port 362so that a needle disposed within the access port engages the stopper 364to withdraw the reconstitution fluid into the tube. After the detectionvial 360 is removed from the access port 362, the stopper 364 isconfigured to seal the detection vial to maintain the vacuum therein.Moreover, the bladder 352 is configured to automatically refill suchthat the reconstitution station 350 is always primed.

Alternatively, FIG. 94 illustrates a reconstitution station 375 thatincludes a rotary valve 376, according to another embodiment. In thisregard, the reconstitution fluid is stored in reservoir 378 and isgravity fed into a second reservoir or bladder. When the rotary valve376 is in a “closed” position, no fluid can escape from the access port380 due to leakage from the bladder. To transfer fluid, the rotary valve376 may be rotated to an “open” position. A detection vial 382 isinserted within the access port 380 whereby fluid can then be removedfrom the bladder when the stopper engages a needle disposed within theaccess port. Rotating the valve 376 to the open position also closes offthe gravity line feeding into the bladder. When the valve 376 is rotatedback to the closed position, the bladder is able to be automaticallyrefilled. The rotary valve 376 may be operated by a knob or othersuitable mechanism for opening and closing the valve, although a rotaryaction is not required in order for effecting such opening and closingof the valve (e.g., a valve actuated through linear motion).

FIG. 95 depicts a reconstitution station 400 that includes a syringe 402in fluid communication with a rotary valve 404, according to anotherembodiment of the present invention. Unlike the prior embodiments, thereconstitution station 400 is not gravity fed, such that thereconstitution fluid is withdrawn from the reservoir 406 and into thesyringe 402 through actuation of the syringe. In this regard, thesyringe 402 may be configured to withdraw a desired amount ofreconstitution fluid into the syringe or a bladder disposed therein whenthe rotary valve 404 is in a closed position. Once the syringe isfilled, rotating the valve 404 to an open position allows access to thefluid contained within the syringe by inserting a detection vial withinthe access port 408 and engaging the needle disposed in the access port408. Rotating the valve 404 to the open position closes off the linefeeding the bladder from the reservoir 406. Again, it is understood thatthe rotary valve 404 may be any suitable mechanism to facilitate openingand closing of the valve. Likewise, the syringe 402 may be any suitabledevice configured to withdraw a desired amount of reconstitution fluidfrom the reservoir 406.

The portrayed examples demonstrate reconstitution stations requiring noexternal power sources. One skilled in the art can also envision fluidmetering systems which are powered.

III. Representative Microorganisms

Embodiments of the present invention can be used to detect suspectedblood stream infections arising from bacteremia and fungemia. Multipleblood samples typically are collected from separate veins of a subject,e.g., a patient, at different time intervals depending on the symptomsof the subject, e.g., the observation of a fever, or some other initialdiagnosis. A volume of the blood sample, e.g., about 3 mL to about 10 mLfor adults and about 1 mL for pediatric samples, can be disposed into ablood culture growth bottle after collection. Typically for eachcollection cycle, one sample is disposed in a blood culture growthbottle suitable for aerobic organisms and one sample is disposed in ablood culture growth bottle suitable for anaerobic organisms.

Unlike methods known in the art that detect an increase in gasproduction as a measure of microbial growth in blood culture samples,the presently disclosed methods advantageously allow for the detectionof intracellular pathogens (e.g., bacterial, viral). Intracellularmicroorganisms or pathogens grow and reproduce within other cells (e.g.,eukaryotic cells) and therefore, cannot be detected using gas sensorsknown in the art. Representative intracellular microorganisms that canbe detected with the presently disclosed methods include, but are notlimited to, Chlamydia trachomatis and Mycobacterium tuberculosis.

The microorganisms presented in Table 1 are commonly found in subjectsas the cause of bacteremia or septicemia and are ranked in the order inwhich they are found in subjects. Also annotated in Table 2 are thosemicroorganisms which collectively represent 80% of all positive resultsin blood culture samples and those microorganisms which are consideredto be under treated.

TABLE 1 Organisms by Occurrence of Bacterial Species or Group in 2002¹Represents 80% of all BC Under Ranking Bacterial Species or Group %positives Treated 1 Coagulase-negative 42 X X Staphylococcus (includingS. epidermidis) 2 S. aureus 16.5 X X 3 E. faecalis 8.3 X 4 E. coli 7.2 XX 5 K. pneumoniae 3.6 X X 6 E. faecium 3.5 X 7 Streptococci viridansgroup 3.4 8 Psuedomanas aeruginosa 2.5 X 9 S. pneumoniae 2.3 10Enterobacter cloacae 1.9 11 serratia marcescens 1.0 12 Acinetobacterbaumannii 0.9 X 13 Proteus mirabilis 0.9 14 Streptococcus agalactiae 0.815 Klebsiella oxytoca 0/6 16 Enterobacter aerogenes 0.5 17Stenotrophomonas maltophilia 0.3 18 Citrobacter freundii 0.3 19Streptocuuocus pyogenes 0.3 20 Enterococcus avium 0.2 21 Others 3.4Fungi > Yeast X C. albicans ¹Karlowsky, J. A. et al., “Prevalence andantimicrobial susceptibilities of bacteria isolated from blood culturesof hospitalized patients in the United States in 2002,” Annals ofClinical Microbiology and Antimicrobials 3: 7 (2004).

Food, water, cosmetic, pharmaceutical and environmental samples arecommonly screened for microorganisms including, but not limited to,enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichiacoli (EPEC), enterohemorrhagic Escherichia coli (EHEC), enteroinvasiveEscherichia coli (EIEC), enteroaggregative Escherichia coli (EAEC),diffusely adherent Escherichia coli (DAEC), shiga toxin-producingEscherichia coli (STEC), E. coli O157, E. coli O157:H7, E. coli O104, E.coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121 and E.coli O145, Shigella species, Salmonella species, Salmonella bongori,Salmonella enterica, Campylobacter species, Yersinia enterocolitica,Yersinia pseudotuberculosis, Vibrio species, Vibrio cholerae, Listeriaspecies, Listeria monocytogenes, Listeria grayii, Listeria innocua,Listeria ivanovii, Listeria seeligeri, Listeria welshmeri,Staphylococcus species, Coagulase negative Staphylococcus species,Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Clostridiumperfringens, Clostridium botulinum, Clostridium tetani, Clostridiumsporogenes, Cronobacter species, Cronobacter sakazakii (formallyEnterobacter sakazakii), Streptococcus species, S. pyogenes, Micrococcusspecies, Psuedomonas species, P. aeruginosa, P. fluorescens, P. putida,Legionella species, Serratia species, K. pneumoniae, Enterobacterspecies, Alcaligenes species, Achromobacter species, yeast and moldssuch as Aspergillus species, Penicillium species, Acremonium species,Cladosporium species, Fusarium species, Mucor species, Rhizopus species,Stachybotrys species, Trichoderma species, Alternaria species,Geotrichum species, Neurospora species, Rhizomucor species, Rhizopusspecies, Ustilago species, Tolypocladium species, Mizukabi species,Spinellus species, Cladosporium species, Alternaria species, Botrytisspecies, Monilia species, Manoscus species, Mortierella species, Oidiumspecies, Oosproa species, Thamnidium species, Candida species,Saccharomyces species, Trichophyton species.

In addition, these samples are often screened for indicator organismsincluding, but not limited to, coliforms, fecal coliforms, E. coli,Enterobacteriaceae, Enterococcus species, coliphage or bacteriophage.

Additionally, some samples are screened for clinically significantantibiotic resistant strains of microorganisms, including, but notlimited to, Methicillin-resistant S. aureus and Vancomycin-resistantEnterococcus species.

Microorganisms that can be detected according to embodiments of thepresent invention include, but are not limited to, Gram negativebacteria, Gram positive bacteria, acid-fast Gram positive bacteria, andfungi, including yeasts. Representative bacterial and fungalmicroorganisms, i.e., antigens, that are targets for the presentlydisclosed blood culture assays are provided immediately herein below,according to one embodiment of the present invention. As noted elsewhereherein, antibodies having specificity for the antigens presentedimmediately herein below can include but are not limited to, polyclonal,monoclonal, Fab′, Fab″, recombinant antibodies, single chain antibodies(SCA), humanized antibodies, or chimeric antibodies. In all cases, theantibody will have one or more CDRs specific for the antigen listedimmediately herein below. Antibodies are known in the art and arereadily available for selected antigens. In some instances, the antigensare present on the cell surface. In other instances, the antigens aresecreted from the cell and are present in the blood culture media as“free antigen.” In yet other instances, both free and bound antigen canbe measured simultaneously to confirm a bacteremia or fungemia.

Regardless of the diagnostic information sought in the culture vessel, aspecific binding member will often have broad specificity. The specificbinding members may be pan-strain, pan-serogroup, pan-species orpan-genera.

The bacterial cell wall is a complex, semi-rigid structure, whichdefines the shape of the organism, surrounds the underlying fragilecytoplasmic membrane, and protects the bacterial cell from the externalenvironment. The bacterial cell wall is composed of a macromolecularnetwork known as peptidoglycan, comprising carbohydrates andpolypeptides that form a lattice around the bacterial cell. Thebacterial cell wall provides the mechanical stability for the bacterialcell and prevents osmotic lysis. Most relevant to the present invention,it is the chemical composition of the cell wall that is used todifferentiate the major species of bacteria.

The cell walls of different species of bacteria may differ greatly inthickness, structure and composition. However, there are two predominanttypes of bacterial cell wall, and whether a given species of bacteriahas one or the other type of cell wall can generally be determined bythe cell's reaction to certain dyes. Perhaps the most widely-used dyefor staining bacteria is the Gram stain. When stained with this crystalviolet and iodine stain, bacteria which retain the stain are called Grampositive, and those that do not are called Gram negative.

As used herein, by “Gram positive bacteria” is meant a strain, type,species, or genera of bacteria that, when exposed to Gram stain, retainsthe dye and is, thus, stained blue-purple.

As used herein, by “Gram negative bacteria” is meant a strain, type,species, or genera of bacteria that, when exposed to Gram stain does notretain the dye and is, thus, is not stained blue-purple. The ordinarilyskilled practitioner will recognize, of course, that depending on theconcentration of the dye and on the length of exposure, a Gram negativebacteria may pick up a slight amount of Gram stain and become stainedlight blue-purple. However, in comparison to a Gram positive bacteriastained with the same formulation of Gram stain for the same amount oftime, a Gram negative bacteria will be much lighter blue-purple incomparison to a Gram positive bacteria.

Representative Gram negative bacteria include, but are not limited to,bacteria in the Enterobacteriaceae family. Representative Gram negativebacteria in the Enterobacteriaceae family include, but are not limitedto bacteria in the Escherichia genus, such as E. coli species (model).Suitable binding members, e.g., antibodies, having an affinity for Gramnegative bacteria in the Enterobacteriaceae family include, but are notlimited to, those antibodies that specifically bind thelipopolysaccharide (LPS) or outer membrane protein (OMP). The LPSLipid-A component, the LPS O-Region, and the LPS core having inner andouter core regions can serve as suitable antigens for specific bindingmembers that have an affinity for Gram negative bacteria in theEscherichia genus.

Representative members of the Escherichia genus include: E.adecarboxylata, E. albertii, E. blattae, E. coli, E. fergusonfi, E.hermannii, and E. vulneris.

Another representative genus within the Enterobacteriaceae family is theKlebsiella genus, including but not limited to, Klebsiella pneumoniae(model). Suitable binding members, e.g., antibodies, having an affinityfor Gram negative bacteria in the Klebsiella genus include, but are notlimited to, those that specifically bind LPS, capsular polysaccharide(CPS) or K antigens (high molecular weight capsular polysaccharide witha molecular weight of about 50 to about 70 kDa), or OMP.

Representative members of the Klebsiella genus include K. granulomatis,K. mobilis, K. ornithinolytica, K. oxytoca, K. ozaenae, K. planticola,K. pneumoniae, K. rhinoscleromatis, K. singaporensis, K. terrigena, K.trevisanfi, and K. varricola.

Gram negative bacteria also include bacteria belonging to theChlamydiaceae family. Representative Gram negative bacteria in theChlamydiaceae family include, but are not limited to, bacteria in theChlamydia genus, such as C. trachomatis species (model). Suitablebinding members, e.g., antibodies, having an affinity for Gram negativebacteria in the Chlamydiaceae family include, but are not limited to,those that specifically bind lipopolysaccharide (LPS) or outer membraneprotein (OMP), including major outer membrane protein (MOMP).

Representative members of the Chlamydia genus include: C. muridarum, C.suis, and C. trachomatis.

Suitable Gram negative bacteria can also include those within thePseudomonas genus, including but not limited to P. aeruginosa (model),the Stenotrophomonas genus, including but not limited to, S. maftophilia(model), and the Acinetobacter genus, including but not limited to A.baumannii (model). Suitable antigens that are recognized by specificbinding members with affinity for Gram negative bacteria within thePseudomonas genus include, but are not limited to, LPS, OMP,iron-regulated membrane proteins (IRMP), flagella, mucoidexopolysaccharide (MEP), and outer membrane protein F (OprF). Suitableantigens that are recognized by specific binding members with affinityfor Gram negative bacteria within the Stenotrophomonas genus include,but are not limited to, LPS, flagella, major extracellular protease,OMP, the 30 kDa exposed protein that binds to the IgG Fc, and the 48.5kDa membrane protein. Suitable antigens that are recognized by specificbinding members with affinity for Gram negative bacteria within theAcinetobacter genus include, but are not limited to, LPS, LPS withD-rhamos, Bap (biofilm associated factor), capsular polysaccharide(CPS), and OMP.

Representative Gram positive bacteria include, but are not limited to,bacteria in the Micrococcaceae family. Gram positive bacteria in theMicrococcaceae family include, but are not limited to, bacteria in theStaphylococcus genus, including S. epidermidis species (model). Suitablebinding members, e.g., antibodies, having an affinity for Gram positivebacteria include, but are not limited to, those that specifically bindto Lipoteichoic Acid (LTA), peptidoglycan, biofilm antigens, including140/200-kDa biofilm antigens and 20-kDa polysaccharide (PS), or Lipid S(glycerophospho-glycolipid). Other suitable binding members that have anaffinity for Gram positive bacteria in the Staphylococcus genus,including but not limited to S. aureus, include those that specificallybind teichoic acid, microbial surface components recognizing adhesionmatrix molecules (MSCRAMMS), iron-responsive surface determinant A(IsdA), the 110 kDa, 98 kDa, and 67 kDa proteins, RNAIII activatingprotein (RAP), target of RNAIII-activating protein (TRAP), alpha toxin,poly-n-succinyl beta-1-6-glucosamine (PNSG), lipase, staphylolysin,FnBPA, FnBPB, immunodominant staphylococcal antigen, capsularpolysaccharide, or the cell surface antigen associated with methycillinresistance.

Representative members of the Staphylococcus genus include: S. aureus,S. auricularis, S. capitis, S. caprae, S. cohnii, S. epidermidis, S.felis, S. haemolyticus, S. hominis, S. intermedius, S. lugdunensis, S.pettenkoferi, S. saprophyticus, S. schleiferi, S. simulans, S. vitulus,S. warneri, and S. xylosus.

Other representative Gram positive bacteria include bacteria in theEnterococcus genus, including but not limited to, E. faecalis (alsoknown as Group D Streptococcus) and E. faecium. Suitable bindingmembers, e.g., antibodies, having an affinity for E. faecalis include,but are not limited to, those that specifically bind to lipoteichoicacid (LTA), collagen binding surface antigen (CNA), aggregationsubstance (AS), capsular polysaccharide, teichoic acid-like capsularpolysaccharide, Esp gene product, Gls24, Epa gene product, Ace (ECMbinder), or peptidoglycan. Suitable binding members, e.g., antibodies,having an affinity for E. faecalis include, but are not limited to,those that specifically bind to ACM protein (collagen binder) or SagAprotein.

Representative acid-fast Gram positive bacteria include, but are notlimited to, bacteria in the Mycobacteriaceae family. Acid-fast Grampositive bacteria in the Mycobacteriaceae family include, but are notlimited to, bacteria in the Mycobacterium genus, such as M. bovis(model) species and M. tuberculosis species (model). Suitable bindingmembers, e.g., antibodies, having an affinity for acid-fast Grampositive bacteria include but are not limited to, those thatspecifically bind to arabinomannon (AM), lipoarabinomannon (LAM) or the38 kDa antigen.

Representative members of the Mycobacterium genus include: M. abscessus,M. africanum, M. agri, M. aichiense, M. alvei, M. arupense, M.asiaticum, M. aubagnense, M. aurum, M. austroafricanum, Mycobacteriumavium complex (MAC), including, M. avium, M. avium paratuberculosis, M.avium silvaticum, M. avium “hominissuis,” M. boenickei, M. bohemicum, M.bolletii, M. botniense, M. bovis, M. branded. M. brisbanense, M. brumae,M. canariasense, M. caprae, M. celatum, M. chelonae, M. chimaera, M.chitae, M. chlorophenolicum, M. chubuense, M. colombiense, M.conceptionense, M. confluentis, M. conspicuum, M. cookii, M. cosmeticum,M. diernhoferi, M. doricum, M. duvalii, M. elephantis, M. fallax, M.farcinogenes, M. flavescens, M. florentinum, M. fluoroanthenivorans, M.fortuitum, M. fortuitum subsp. acetamidolyticum, M. frederiksbergense,M. gadium, M. gastri, M. genavense, M. gilvum, M. goodii, M. gordonae,M. haemophilum, M. hassiacum, M. heckeshornense, M. heidelbergense, M.hiberniae, M. hodleri, M. holsaticum, M. houstonense, M. immunogenum, M.interjectum, M. intermedium, M. intracellulare, M. kansasii, M.komossense, M. kubicae, M. kumamotonense, M. lacus, M. lentiflavum, M.leprae, M. lepraemurium, M. madagascariense, M. mageritense, M.malmoense, M. marinum, M. massiliense, M. microti, M. monacense, M.montefiorense, M. moriokaense, M. mucogenicum, M. murale, M.nebraskense, M. neoaurum, M. neworleansense, M. nonchromogenicum, M.novocastrense, M. obuense, M. palustre, M. parafortuitum, M.parascrofulaceum, M. parmense, M. peregrinum, M. phlei, M. phocaicum, M.pinnipedii, M. porcinum, M. poriferae, M. pseudoshottsii, M. pulveris,M. psychrotolerans, M. pyrenivorans, M. rhodesiae, M. saskatchewanense,M. scrofulaceum, M. senegalense, M. seoulense, M. septicum, M.shimoidei, M. shottsii, M. simiae, M. smegmatis, M. sphagni, M. szulgai,M. terrae, M. thermoresistibile, M. tokaiense, M. triplex, M. triviale,Mycobacterium tuberculosis complex (MTBC), including M. tuberculosis, M.bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. pinnipedii,M. tusciae, M. ulcerans, M. vaccae, M. vanbaalenii, M. wolinskyi, and M.xenopi.

Representative fungi, including yeasts, include, but are not limited to,the Saccharomycetaceae family, including, the Candida genus, such aswith C. albicans (model). Suitable binding members, e.g., antibodies,having an affinity for fungi belonging to the Candida genus include, butare not limited to, those that specifically bind to mannan,phosphomannan, annoprotein 58 (mp58), galactomannan, Beta-D-Glucan,metalloabinitol, Cell Wall-associated glyceraldehyde-3-phosphatedehydrogenase, Enolase-(47/48 kDa), Secreted-Aspartyl-Proteinase (SAP),or heat shock protein 90 (HSP-90).

Representative members of the Candida genus include: C. aaseri, C.albicans, C. amapae, C. anatomiae, C. ancudensis, C. antillancae, C.apicola, C. apis, C. atlantica, C. atmosphaerica, C. auringiensis, C.austromarina, C. azyma, C. beechii, C. bertae, C. berthetii, C. blankii,C. boidinii, C. boleticola, C. bombi, C. bombicola, C. buinensis, C.butyri, C. cantarellii, C. caseinolytica. C. casteffii, C. castrensis,C. catenulata, C. chilensis, C. chiropterorum, C. chodatii, C. ciferrii,C. coipomoensis, C. conglobata, C. cylindracea, C. dendrica, C.dendronema, C. deserticola, C. diddensiae, C. diversa, C. drimydis, C.dubliniensis, C. edax, C. entomophila, C. ergastensis, C. emobii, C.ethanolica, C. euphorbiae, C. euphorbiiphila, C. fabianii, C. famata, C.famata var. famata, C. famata var. flareri, C. fennica, C.fermenticarens, C. firmetaria, C. floricola, C. fluviatilis, C.freyschussii, C. friedrichii, C. fructus, C. galacta, C. geochares, C.glabrata, C. glaebosa, C. glucosophila, C. gropengiesseri, C.guilliermondii, C. guilliermondii var. guilliermondii, C. guilliermondiivar. membranaefaciens, C. haemulonii, C. homilentoma, C. humilis, C.incommunis, C. inconspicua, C. insectalens, C. insectamans, C.insectorum, C. intermedia, C. ishiwadae, C. karawaiewii, C. kefyr, C.krissii, C. kruisii, C. krusei, C. lactis-condensi, C. laureliae, C.lipolytica, C. llanquihuensis, C. lodderae, C. lusitaniae, C.lyxosophila, C. magnoliae, C. maltosa, C. maris, C. maritima, C.melibiosica, C. membranifaciens, C. mesenterica, C. methanosorbosa, C.milleri, C. mogii, C. montana, C. multigemmis, C. musae, C. naeodendra,C. natalensis, C. nemodendra, C. norvegensis, C. norvegica, C.odintsovae, C. oleophila, C. oregonensis, C. ovalis, C. palmioleophila.C. paludigena, C. parapsilosis, C. pararugosa, C. pelliculosa, C.peltata, C. petrohuensis, C. pignaliae, C. pini, C. populi, C.pseudointermedia, C. pseudolambica, C. psychrophila, C. pulcherrima, C.quercitrusa, C. quercuum, C. railenensis, C. reukaufii, C. rhagii, C.robusta, C. rugopelliculosa, C. rugosa, C. saitoana, C. sake, C. salida,C. salmanticensis, C. santamariae, C. santjacobensis, C. savonica, C.schatavii, C. sequanensis, C. shehatae, C. shehatae var. Insectosa, C.shehatae var. lignosa, C. shehatae var. shehatae, C. silvae, C.silvanorum, C. silvatica, C. silvicultrix, C. solani, C. sonorensis, C.sophiae-reginae, C. sorbophila, C. sorbosa, C. sorboxylosa, C.spandovensis, C. stellata, C. succiphila, C. suecica, C. tanzawaensis,C. tapae, C. techellsii, C. tenuis, C. torresii, C. tropicalis, C.tsuchiyae, C. utilis, C. vaccinii, C. valdiviana, C. valida, C.vanderwaltii, C. vartiovaarae, C. versatilis, C. vini, C. viswanathii,C. wickerhamii, C. xestobii, and C. zeylanoides.

Therapeutic antibodies such as Aurograb™ with specificity for theMethicillin-resistant S. aureus (MRSA) strains also can be used oncapture or indicator surfaces. Likewise the therapeutic monoclonalantibody (mab) Myograb™ (Efungumab) with a specificity for the Heatshock Protein HSP90 can be used for detection of C. albicans.

The presently disclosed SERS-active indicator particles can bedistinguished from the many other optically active materials that can bepresent in a culture environment, such as components of culture mediaused to support growth, whole blood, SPS anticoagulant, foodparticulates, and additives. Further, the specific SERS-active indicatorparticles exhibit the necessary signal intensity to allow detection ofsmall quantities of bacterial cells. Additionally, a variety ofSERS-active indicator particles, each having a unique SERS signature,allow blood culture samples to be interrogated for any one of aplurality of microorganisms (e.g., twenty) that can typically be foundin mammalian, e.g., human, blood. In such embodiments, the detection ofeach particular microorganism can occur simultaneously, which isreferred to herein as a “multiplex assay.”

According to one embodiment, for example, blood culture, the primarytargets for the presently disclosed multiplex assays include:Coagulase-negative Staphylococci, S. aureus, E. faecalis, E. coli, K.pneumoniae, E. faecium, Viridans group Streptococci, Pseudomonasaeruginosa, S. pneumoniae, Enterobacter cloacae, Serratia marcescens,Acinetobacter baumannii, Proteus mirabilis, Streptococcus agalactie,Klebsiella oxytoca, Enterobacter aerogenes, Stenotrophomonasmaltophilia, Citrobacter freundii, Streptococcus pyogenes, andEnterococcus avium. Such multiple targets can be, in some embodiments,be simultaneously detected by a presently disclosed multiplex assay.

IV. Representative Culture Media

Representative culture media suitable for use with embodiments of thepresent invention are provided immediately herein below. One of ordinaryskill in the art would recognize that the presently disclosedformulations can be modified to meet specific performance requirements.Additionally, these formulations, depending on the particularapplication, can have disposed therein, CO₂, O₂, N₂, and combinationsthereof, to create an environment suitable for aerobic, anaerobic, ormicroaerophilic growth. Optionally, some culture media containadsorbents to isolate, i.e., remove, from the culture medium,interferents, such as antibiotics or immune elements that can be presentin a subject's blood sample or metabolites produced during culture. See,e.g., U.S. Pat. No. 5,624,814, which is incorporated herein by referencein its entirety. For example, the BD BACTEC™ Media Plus Anaerobic/F, BDBACTEC™ Plus Aerobic/F, and BD BACTEC™ PEDS Plus/F, each of which isavailable from Becton, Dickinson, and Company, Franklin Lakes, N.J., allcontain resins for isolating antibiotics that otherwise can inhibitmicrobial growth in the blood culture medium. The resins aresubstantially larger in diameter than any component of blood and aremore rigid than the mammalian cells found in blood. Another example of aculture absorbent is the precipitated calcium carbonate (1%-2.5% w/v)found in various Tetrathionate Broth formulations used for selectivelyculturing Salmonella in food and environmental samples. The calciumcarbonate particulates neutralize the sulfuric acid produced by thereduction of tetrathionate by growing Salmonella.

A. BD BACTEC™ Myco/F Lytic Culture Vials

BD BACTEC™ Myco/F Lytic Culture Vials support the growth and detectionof aerobic microorganisms. More particularly, BD BACTEC™ Myco/F LyticCulture Vials are non-selective culture media to be used as an adjunctto aerobic blood culture media for the recovery of mycobacteria fromblood specimens and yeast and fungi from blood and sterile body fluids.

Mycobacterium tuberculosis (MTB) and mycobacteria other thantuberculosis (MOTT), especially Mycobacterium avium complex (MAC), havebecome resurgent. From 1985 to 1992, the number of MTB cases reportedincreased 18%. Between 1981 and 1987, AIDS case surveillances indicatedthat 5.5% of the patients with AIDS had disseminated nontuberculousmycobacterial infections, e.g., MAC. By 1990, the increased cases ofdisseminated nontuberculous mycobacterial infections had resulted in acumulative incidence of 7.6%. The incidence of fungemia also hassteadily increased since the early 1980s. These increases haveheightened the need for effective diagnostic procedures for fungemia andmycobacteremia.

Components of the presently disclosed formulations can include, but arenot limited to, ferric ammonium citrate or an equivalent that providesan iron source for specific strains of mycobacteria and fungi, saponinor an equivalent blood lysing agent, and specific proteins and sugars toprovide nutritional supplements.

B. BD BACTEC™ 12B Mycobacteria Culture Vials Middlebrook 7H12

The qualitative BACTEC™ 12B Mycobacteria Medium can be used for theculture and recovery of mycobacteria from clinical specimens, sputum,gastric, urine, tissue, mucopurulent specimens, other body fluids andother respiratory secretions, differentiation of the Mycobacteriumtuberculosis complex from other mycobacteria, and drug susceptibilitytesting of M. tuberculosis.

C. BACTEC™ LYTIC/10 Anaerobic/F Culture Vials

The BACTEC™ LYTIC/10 Anaerobic/F The BACTEC™ LYTIC/10 Anaerobic/F mediumis also suitable for embodiments of the present invention.

D. BACTEC™ Plus Aerobic/F* and Plus Anaerobic/F* Culture VialsSoybean-Casein Digest Broth

BACTEC™ Plus Aerobic/F and Plus Anaerobic/F media provide a qualitativeprocedure for the culture and recovery of microorganisms (bacteria andyeast) from blood and have been formulated to allow the addition of upto 10 mL of blood. The addition of these larger sample volumes resultsin overall higher detection rates and earlier times to detection.

E. BD BACTEC™ Standard Anaerobic/F Culture Vials Soybean-Casein Digest

BD BACTEC™ Standard Anaerobic/F Culture Vials Soybean-Casein Digestbroth provides a qualitative procedure for the culture and recovery ofanaerobic microorganisms from blood.

F. BD BACTEC™ PEDS PLUS™/F Culture Vials

BACTEC™ culture vials type PEDS PLUS™/F (enriched Soybean-Casein Digestbroth with CO₂) are intended for use with aerobic cultures and providefor the culture and recovery of aerobic microorganisms (mainly bacteriaand yeast) from pediatric and other blood specimens which are generallyless than 3 mL in volume.

G. Standard/10 Aerobic/F Culture Vials

BACTEC™ Standard/10 Aerobic/F culture vials (enriched Soybean-CaseinDigest broth with CO₂) are intended for use in aerobic blood culturesand provide for the culture and recovery of aerobic microorganisms(bacteria and yeast) from blood.

H. BacT/ALERT™ Culture Vials

BacT/ALERT™ FAN, BacT/ALERT™ FN, and BacT/ALERT™ SN culture vials(bioMérieux, Durham, N.C.) are intended for use in anaerobic bloodcultures and provide for the culture and recovery of anaerobicmicroorganisms (bacteria and yeast) from blood.

I. Selective E. coli Culture Media

Modified Buffered Peptone water with pyruvate (mBPWp) andAcriflavin-Cefsulodin-Vancomycin (ACV) Supplement is a media prescribedby the FDA Bacteriological Analytical Manual (BAM) for enriching samplesfor the detection of diarrheagenic Escherichia coli.

J. Selective Listeria Culture Media

Frasier Broth Base and Fraser Broth Supplement are used to selectivelyenrich and detect Listeria species. The USDA Microbiological LaboratoryGuidebook (MLG) recommends the use of Fraser Broth when testing for L.monocytogenes in red meat, poultry, egg and environmental samples (USDAMLG Chapter 8.07, revised Aug. 3, 2009).

K. Selective Salmonella Culture Media

Tetrathionate Base Broth, Hajna is a media designed for the selectiveenrichment of Salmonella. Tetrathionate is generated by the addition ofiodine and potassium iodide just prior to enrichment. The USDAMicrobiological Laboratory Manual stipulates this broth for theselective enrichment of Salmonella in meat, poultry, pasteurized egg andcatfish products (USDA MLG Chapter 4.05, revised Jan. 20, 2011).

L. Salmonella Culture Media

In addition to the culture media listed above, there are several brothscommonly known in the art to culture or sustain Salmonella, including,but not limited to, Brain Heart Infusion Broth, Brilliant Green SulfaEnrichment (BD Difco™), modified Brilliant Green Broth (BD Difco™),Buffered Peptone Water (BD Difco™), Buffered Peptone Casein Water (BDDifco™), Dey-Engly Broth (BD Difco™), EE Broth Mossel Enrichment (BDDifco™), Gram Negative Broth (BD Difco™), Gram Negative Broth Hajna (BDDifco™), Lactose Broth (BD Difco™), Letheen Broth (BD Difco™), LysineDecarboxylase Broth, M Broth (BD Difco™), Malonate Broth (BD Difco™),MR-VP Broth, Nutrient Broth, One Broth-Salmonella (Oxoid), Phenol RedCarbohydrate Broth (BD BBL™), Potassium Cyanide Broth, PurpleCarbohydrate Broth (BD BBL™), RapidChek® Salmonella primary media(SDIX), RapidChek® SELECT™ Salmonella primary with supplement (SDIX),RapidChek® SELECT™ Salmonella secondary media (SDIX),Rappaport-Vassiliadis Medium, modified Rappaport-Vassiliadis Medium,Rappaport-Vassiliadis R10 Broth (BD Difco™), Rappaport-VassiliadisSalmonella (RVS) Soy Broth (BD Difco™), Rappaport-Vassiliadis SoyaPeptone Broth, Selenite Broth (BD Difco™), Selenite-F Broth (BD BBL™),Selenite Cystine Broth (BD Difco™), Tetrathionate Broth, Tetrathionate(Hajna) Broth, Tryptone Broth, Tripticase Soy Broth, Tripticase SoyBroth with ferrous sulfate, Universal Preenrichment Broth, UniversalPreenrichment Broth without ferric ammonium citrate, and Urea Broth.

M. Listeria Culture Media

In addition to the culture media listed above, there are several brothscommonly known in the art to culture or sustain Listeria, including, butnot limited to, Brain Heart Infusion (BHI) Broth, Buffered ListeriaEnrichment Broth (BLEB), Nutrient Broth, Purple carbohydratefermentation broth base (M130¹⁵), containing 0.5% solutions of dextrose,esculin, maltose, rhamnose, mannitol, and xylose, SIM medium, Trypticasesoy broth with 0.6% yeast extract, Tryptose Broth, Modified Universityof Vermont (UVM) Broth, Morpholinepropanesulfonic acid-buffered Listeriaenrichment broth (MOPS-BLEB), Demi-Frasier, Fraser broth, Listeriaenrichment broth (BD Difco™, Oxoid), One Broth-Listeria (Oxoid),RapidChek® Listeria media with supplement (SDIX) and RapidChek® ListeriaF.A.S.T.™ media (SDIX).

V. Representative Samples

The amount of one or more microorganisms present in a sample under testcan be represented as a concentration. The concentration can beexpressed as a qualitative value, for example, as a negative- orpositive-type result, e.g., a “YES” or “NO” response, indicating thepresence or absence of a microorganism, or as a quantitative value.Further, the concentration of a given microorganism in a culture samplecan be reported as a relative quantity or an absolute quantity, e.g., asa “quantitative value.”

The quantity (i.e., concentration) of a microorganism can be equal tozero, indicating the absence of the particular analyte sought or thatthe concentration of the particular analyte is below the detectionlimits of the assay. The quantity measured can be the signal, e.g., aSERS signal, without any additional measurements or manipulations.Alternatively, the quantity measured can be expressed as a difference,percentage or ratio of the measured value of the particularmicroorganism to a measured value of another compound including, but notlimited to, a standard or another microorganism. The difference can benegative, indicating a decrease in the amount of measuredmicroorganism(s). The quantities also can be expressed as a differenceor ratio of the microorganism(s) to itself, measured at a differentpoint in time. The quantities of microorganism can be determineddirectly from a generated signal, or the generated signal can be used inan algorithm, with the algorithm designed to correlate the value of thegenerated signals to the quantity of microorganism(s) in the sample. Asdiscussed above, embodiments of the present invention are amenable foruse with devices capable of measuring the concentrations of one or moremicroorganisms in real time.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The following Examples are offered by way ofillustration and not by way of limitation.

Example 1 Effect of SERS HNW Reagents on Time to Detection for E. Coli

FIG. 54 shows the result of an experiment in which time to detection ofE. coli growth was compared for blood culture samples with and withoutthe SERS HNW reagents suitable for use in the various embodiments of theinvention.

In this example, unconjugated SERS-active indicator particles (SERS 440tags) and unconjugated magnetic capture particles (Dynal® beads) weresterilized by washing with 70% ethanol. The sterilized SERS-activeindicator particles and magnetic capture particles were then added toBACTEC™ Standard/10 Aerobic/F Medium bottles inoculated with E. coli.The time to detection for E. coli growth by a BACTEC™ 9050 sensor wascompared for bottles with and without the HNW assay reagents. BACTEC™bottles without E. coli but with and without the HNW assay reagents wereincluded as negative controls. As can be seen, the BACTEC™time-to-detection was unaffected by the presence of the SERS-activeindicator particles and magnetic particles in this experiment. Thus theSERS HNW assay reagents do not significantly impact the ability of amicroorganism to grow.

Example 2 Repeated Pelleting is Compatible with Microorganism Growth

FIG. 55 shows the result of an experiment in which SalmonellaTyphimurium growth was monitored during the course of an experiment todetermine if pelleting negatively affects organism growth.

In this example, S. Typhimurium (ATCC 14028) was grown in an overnightculture in SDIX Salmonella Select Primary Media with supplement at 42°C. A 1:100 dilution was made into SDIX Salmonella Secondary Media. Thestarting inoculation in secondary media was determined to be 1.8×10⁷cfu/ml by plate count on Nutrient agar plates. The inoculated secondarymedia was then put into multiple tubes, all containing SERS tags andmagnetic particles conjugated to SDIX Salmonella antibodies. The tubeswere placed in the system 150 (see FIG. 24) for monitoring during growthat 42° C. The tubes were pelleted and interrogated every 0.5 hour duringgrowth. In this experiment, tubes were removed from the instrument after1, 3, 6, 9 and 11 pelleting and reading cycles. These tubes wereenumerated by plating dilutions onto Nutrient agar plates. As can beseen, the growth of S. Typhimurium is not compromised by the presence ofSERS tags and magnetic particles, nor is it compromised by repeatedpelleting and interrogation of the pellet by the laser.

Example 3 Effect of Adjusting Pelleting Frequency

In an experiment examining the effect of repeated pelleting onmicro-organism growth and assay performance, a single colony ofSalmonella Kentucky (ATCC 9263) was picked from a BD BBL™ Nutrient Agarstreak plate and cultured overnight at 42° C. in 6 mL SDIX RapidChek®Salmonella SELECT™ primary culture media with 60 μL phage supplement.Following the primary culture, 5 mL of a secondary culture medium wasprepared, consisting of 90% secondary and 10% primary SDIX RapidChek®Salmonella SELECT™ media. In parallel, a 1:100 dilution of primaryculture into the primary medium was prepared, and 125 μL of thatdilution was inoculated into a BD MGIT™ tube containing the 5 mL ofsecondary medium, 16 μL of SERS tags, and 20 μL of magnetic beads. Theresulting dilution of 1:4000 from the final concentration of the primaryculture yielded an approximate inoculation concentration of 2.5×10⁵CFU/mL. The tubes were then put into one of two carousel-based systems(see e.g., FIG. 24) for 24 hours at 42° C. at a linear agitation speedof ˜1 Hz. All experimental parameters except read frequency were keptthe same between the two instruments. The read frequency for eachinstrument was set to either 5 or 2 reads/hour.

FIG. 56 shows a representative set of data from the experiment. The datafrom the two systems are shown, with intensity axes scaled forcomparison. (Note that the absolute intensities of tag weights betweeninstruments should not be compared due to differences in opticalefficiencies.) The shapes of the growth curves are nearly identical,indicating that the increased number of cycles of pellet formation,measurement, and dispersal did not impede growth or detection. The onlysignificant difference is that the curve with more frequent readingsprovides better resolution on the growth kinetics.

Example 4 Effect of Relative Motion of Sample Tube and Magnets

Reproducible pellet formation is a critical step to achieve reproducibleassay signal. This example pertains to two distinct ways to form apellet. In the first (fixed magnet), the magnet is held fixed in place,while the tube is moved over the magnet for the full extent of theagitation throw. In the second preferred configuration (coupled), shownin FIG. 49, the magnet moves along with the tube. In a series ofexperiments, SERS tags were covalently linked to tosyl-activatedmagnetic particles to form a SERS-magnetic bead pre-complex (PC).Pre-complexed beads are prepared by covalent linkage of SERS particlesto Dynabeads® M-280 Tosyl-activated magnetic particles through reactionof thiol(-SH) groups on the SERS surface with tosyl (Tos) groups on thesurface of the magnetic particles PC acts as a model system for pelletformation testing where the pellet can be interrogated for SERS signal.Pellet formation of PC in water as well as in a commercial secondarymedia for Salmonella (SDIX RapidChek® Salmonella SELECT™) was comparedfor the fixed magnet and coupled geometries. These tests were performedusing a flat-bed system configuration (see e.g., FIG. 25).

FIG. 57 shows an image of PC in water after pelleting with a fixedmagnet. PC in water was pelleted for 1.5 minutes by moving the tube overa fixed bar magnet at 0.5 Hz agitation frequency and 25 mm throw.Agitation was stopped and the magnet was allowed to persist for 30seconds before moving the bar away from the tubes. As shown in FIG. 57,a single pellet was formed. This image highlights the ability to dragthe magnetic complexes with the magnet through water.

In contrast, FIGS. 58A and 58B show PC pellet formation in SDIXSalmonella secondary media using a fixed magnet and two differentagitation frequencies. PC in SDIX secondary media was pelleted for 3minutes by moving the tube over a fixed bar magnet at either 2 Hz (21A)or 0.5 Hz (21B) agitation frequency and 25 mm throw. Agitation wasstopped and the magnet was allowed to persist for 30 seconds beforemoving the magnet bar away from the tubes. As shown in FIG. 58A, twopellets of magnetic complexes were pulled to the bottom of the tube,located at the limits of the relative motion between the tube and themagnet. For the slower agitation (FIG. 58B), two pellets were formed atthe ends of the magnet travel along with an ill-defined line connectingthe pellets. As reproducible SERS signal is best obtained with a dense,reproducibly placed pellet, FIGS. 58A and 58B highlight thedisadvantages of the fixed magnet configuration, which appears unable todrag the magnetic beads through SDIX Salmonella secondary media,presumably due to the solid particulates present in this media.

FIG. 59A shows a preferred embodiment using the coupled magnetconfiguration. PC in SDIX Salmonella secondary media was pelleted for1.5 minutes by moving the tube coupled with a bar magnet at 1.5 Hzagitation frequency and 25 mm throw. Agitation was stopped and themagnet was allowed to persist for 30 seconds before moving the barmagnet away from the tubes. A single dense pellet was formed where thebar magnet contacts the sample tube. Compared to the pellets formedusing the fixed magnet configuration, the pellet formed using coupledmagnets is more compact and dense, as shown in FIG. 59A. FIG. 59B showsa similar experiment with a coupled magnet, only using a 0.8 Hzagitation frequency. Although a single pellet was formed, settled mediainterferes with the ability to pull a dense pellet, as evidenced by thediffuse particles in the center of the pellet.

The results illustrated in FIGS. 57-59 show that for a throw of 25 mm,the fixed magnet pelleting approach failed to form a single dense pelletin the presence of SDIX Salmonella secondary media using a variety ofagitation frequencies. This media contains solid particulates thatsettle rapidly and interfere with the ability of the magnet to drag themagnetic complexes along the bottom of the tube. Although fast agitationwill keep solid media from settling, two pellets are formed at thelimits of the relative motion between the tube and the magnet. Asagitation slows to a stop, these pellets cannot be dragged through themedia to form a single pellet. Because the magnetic complexes can bedragged through water, the fixed magnet approach can be used to pelletPC in water.

The coupled magnet pelleting approach forms a single dense pellet in thepresence of SDIX Salmonella secondary media at a variety of agitationfrequencies. Coupling magnets to the tube for pelleting does not requiremagnetic complexes to drag along the bottom of the tube because they arepulled to a common point to form a single pellet.

Using coupled magnets, fast agitation forms a denser pellet compared toslow agitation. This is likely due to the solid media settling usingslow agitation and interfering with pellet formation. Using fastagitation, the solid is suspended in solution and magnetic complexes canbe pulled into a pellet with less interference from the media.

Example 5 Singleplex Detection of C. albicans in Human Blood

FIG. 60 shows an example of detection and identification ofmicroorganisms within a blood culture sample (spiked blood) for asingleplex format. In this experiment, Candida albicans (ATCC 10231) wasgrown in an overnight culture in Sabouraud Dextrose Broth from a singlecolony at 30° C. in a shaking culture. The culture was diluted down andinoculated into human blood at 3 cfu/ml or 0 cfu/ml as a negativecontrol. Positive and negative samples were inoculated into BACTEC™ Std10 Aerobic/F bottles without detection reagents as well as into tubescontaining BACTEC™ Std 10 Aerobic/F media and the detection reagents(SERS tags and magnetic particles conjugated with Virostat 6411anti-Candida albicans antibody). The overall blood to media ratio was1:8. The inocula were plated on BBL™ CHROMagar™ for enumeration.Detection tubes were inserted into the carousel system 150 (see FIG. 24)and BACTEC™ bottles were inserted into the BACTEC™ FX instrument forreal time monitoring during growth at 35° C. The positive SERS tube wasdetected at 18 hours and the BACTEC™ bottle was positive at 30 hours.SERS signal provides detection and ID at least 12 hours before BACTEC™FX for this singleplex assay in human blood.

Example 6 Detection of C. albicans in a 4-Pex Assay Format

FIG. 61 shows an example of detection and identification ofmicroorganisms within a blood culture sample (spiked blood) for amultiplex format. In this 4-plex assay for the detection of C. albicans,E. coli 0157, K. pneumoniae, and S. aureus, SERS tags with four distinctRaman reporters were each conjugated to antibodies for one of the fourorganisms. (Antibodies conjugated to SERS tags Virostat 6411 polyclonalanti-C. albicans, Biodesign MAV119-499 monoclonal anti-E. coli O157:H7,Biodesign C55573M monoclonal anti-Staphylococcus and Santa CruzBiotechnology sc-80861 monoclonal anti-K. pneumoniae). All four SERS tagtypes were present in the assay mixture, along with magnetic beadsconjugated with capture antibodies for the four microorganisms. Themagnetic beads present in the assay were a pool of magnetic beadsconsisting of Dynal® anti-E. coli 0157 magnetic particles (LifeTechnologies catalog #710-03), Dynal® M280 beads conjugated to Virostat6411 polyclonal anti-C. albicans, Dynal® M280 beads conjugated withBiodesign C55573M monoclonal anti-Staphylococcus, and Dynal® M280 beadconjugated with Affinity Bioreagents Pa.1-7226 polyclonal anti-K.pneumoniae.

In the experiment depicted in FIG. 61, C. albicans (ATCC 10231) wasgrown in an overnight culture in Sabouraud Dextrose Broth from a singlecolony at 30° C. in a shaking culture. The culture was diluted down andinoculated into human blood at 3 cfu/ml or 0 cfu/ml as a negativecontrol. Positive and negative samples were inoculated into BACTEC™ Std10 Aerobic/F bottles as well as sample tubes containing BACTEC™ Std 10Aerobic/F media with the detection reagents. The blood to media ratio inthe final sample was 1:8. The C. albicans inocula were plated on BBL™CHROMagar™ for enumeration. The sample tubes containing the SERSreagents were inserted into a carousel system (see FIG. 24), while theBACTEC™ bottles without detection reagents were inserted into theBACTEC™ FX instrument for real time monitoring during growth at 35° C.

C. albicans was detected by SERS at 16.6 hours, while the BACTEC™ gassensor gave positive detection at 28 hours. Furthermore, detection bySERS was accompanied by identification of the microorganism as C.albicans, whereas the BACTEC™ instrument provided no identificationinformation. As can be seen in FIG. 61, the detection of C. albicans bySERS in a multiplexed format resulted in no significant SERS signal fromthe other (non-C. albicans) SERS tags.

Example 7 Detection of E. coli and S. epidermis Coninfection in RabbitBlood

FIG. 62 shows an example of multiplexed detection and identification ofmicroorganisms within a blood culture sample (spiked blood) for a modelco-infection. E. coli O157:H7 (ATCC 700728) and S. epidermidis (ATCC55133) were separately grown in overnight cultures in BD Nutrient Brothfrom a single colony at 37° C. in a shaking culture. The cultures werediluted down and co-inoculated into rabbit blood at 2.6 cfu/ml for E.coli O157:H7 and 12.5 cfu/ml for S. epidermidis. Positive and negativesamples were inoculated into BACTEC™ Std 10 Aerobic/F bottles (no SERSreagents) as well as tubes containing BACTEC™ Std 10 Aerobic/F media andthe detection reagents described in Example 6. (SERS tags conjugated toVirostat 6411, Biodesign MAV119-499, Biodesign C55573M and Santa CruzBiotechnology sc-80861, as well as Dynal® anti-E. coli O157 magneticparticles and Dynal® M280 particles conjugated to Virostat 6411,Biodesign C55573M and Affinity Bioreagents PA1-7226.) The blood wasdiluted 1:8 in BACTEC™ media. The inocula were plated on BBL™ CHROMagar™for enumeration. Detection tubes containing the SERS reagents wereinserted into the carousel system 150 (see FIG. 24), while BACTEC™bottles without SERS reagents were inserted into the BACTEC™ FXinstrument for real time monitoring during growth at 35° C. E. coliO157:H7 was detected and identified by SERS at 7.9 hours, while S.epidermidis was detected and identified by SERS at 11.4 hours. TheBACTEC™ bottle was positive at 10.4 hours, but provided no level ofidentification.

Example 8 Real-Time SERS Detection in Samples Containing Particulates

In this example, E. coli O157:H7 (ATCC 700728) was thawed from aglycerol stock and inoculated into rabbit blood diluted into BACTEC™Plus Aerobic/F Media at a ratio of 1:8. BACTEC™ Plus Aerobic/F Mediacontains resin particles (17% w/v) to enhance the recovery of organismswithout the need for special processing. The inoculated blood plus mediawas enumerated by plate counts to confirm an inoculation of 5 cfu/ml.The sample was placed in three replicate tubes containing SERS andmagnetic bead conjugates (Biodesign MAV119-499 and G5V119-500antibodies). Detection tubes were inserted into the carousel system 150(see FIG. 24) for real time monitoring during growth at 35° C. Resultsare shown in FIG. 63. The carousel system was able to efficiently formand interrogate a pellet in the presence of the resin.

Example 9 Detection in Large Volumes

The agitation provided during pelleting allows magnetic beads to becaptured efficiently, even in large sample volumes or at low magneticbead concentrations.

In one example, assays were conducted with SERS and magnetic particlereagent volumes held constant, while varying sample volumes to achieve arange of reagent concentrations. Samples of 5, 10, 20, 30, 40, and 50 mLof a 1:10 dilution of rabbit blood in BD BACTEC™ Standard 10 Aerobic/Fblood culture medium were tested in 50 mL Falcon™ tubes on acarousel-based assay system modified for large sample volumes (see e.g.,FIG. 24). E. coli O157 was thawed from a frozen stock and spiked intoeach sample at 10⁴ cfu/mL. Over a course of six days, each volume wastested in triplicate, with only one sample of a given volume tested perday.

In each tube, a master mix typically used for 5 mL samples was createdby combining 125 μL of SERS tags and 80 μL of magnetic particles in 795μL of 1:10 blood and media. The resulting 1 mL master mix was added toeach test sample. SERS tags conjugated with Biodesign MAV119-499 anti-E.coli antibodies, and Dynabeads® Anti-E. coli O157 (710-04) magneticparticles from Life Technologies™, were used.

Samples were placed in a carousel-based assay system (see e.g., FIG. 24)at 35° C., with pelleting for 60 sec, an incident laser power of 50 mW,a 5 sec CCD integration time, rocker operating at ˜0.5 cycles/sec, and aread frequency of 5/hour.

Results for a representative sample of each volume are shown in FIG. 64.Although the signal strength is reduced with lower reagentconcentrations, the system is able to effectively form pellets anddetect growth even at a concentration 10× lower than the standard. Ascan be seen, the assay effectively detects growth for a variety ofvolumes.

Example 10 Failure to Pellet Using Fast Agitation in Carousel System

In the carousel system (see e.g., FIG. 24), the samples are agitatedwhile the magnetic pellet is being formed to ensure that magneticcomplexes from the full fluid volume pass through the localized magneticfield. A camera captures images of the pellet during laser interrogationto monitor pellet formation throughout the assay.

FIG. 65A shows an example in which a pellet fails to form within a fewhours of secondary enrichment of 10⁷ CFU/mL Salmonella Typhimurium (ATCC14028) at fast agitation frequencies in the carousel system 150 (seeFIG. 24). Salmonella Typhimurium (ATCC 14028) was cultured overnight inSDIX RapidChek® Salmonella SELECT™ Primary Media with supplement at 42°C. A 1:100 dilution of the culture with SDIX Salmonella Secondary Mediawas inoculated into a secondary container with conjugated magneticparticles and SERS tags and placed into the carousel system 150. Thestarting inoculation in secondary media was determined to be 1×10⁷CFU/mL by plate count on Nutrient agar plates. The instrument read 2times per hour, pelleted for 30 seconds, and agitated at 2 Hz with 25 mmthrow. FIG. 65A shows the resulting SERS curve with images captured atvarious times during secondary enrichment. The first pellet imagecontains a yellow circle to highlight the area where the pellet shouldform. This figure shows the pellet grows in size by 2 hours and fails toform near 3 hours of secondary enrichment time.

For very high loads of Salmonella, the pellet becomes particularly largebecause there is a lot of pathogen present in the pellet. When agitationis too fast, the magnetic field is unable to overcome the fluiddynamics, and the pellet fails to form.

FIG. 65B shows the SERS curve and corresponding images during secondaryenrichment for a negative sample (conjugated magnetic beads andconjugated SERS tags in media). This data shows a consistently low Ramansignal and consistent pellet size during the assay.

By slowing the agitation frequency to 1 Hz during secondary enrichmentof 10⁷ CFU/mL of Salmonella Typhimurium (ATCC 14028), the pelletconsistently formed throughout the assay. Salmonella Typhimurium (ATCC14028) was cultured overnight in SDIX RapidChek® Salmonella SELECT™Primary Media with supplement at 42° C. A 1:100 dilution of the culturewith SDIX Salmonella Secondary Media was inoculated into a secondarycontainer with conjugated magnetic particles and SERS tags and placedinto the carousel system 150. The starting inoculation in secondarymedia was determined to be 1×10⁷ CFU/mL by plate count on Nutrient agarplates. The instrument read 2 times per hour, pelleted for 30 seconds,and agitated at 1 Hz with 25 mm throw. FIG. 65C shows the resulting SERScurve with images captured at various times during secondary enrichment.The first pellet image contains a yellow circle to highlight the areawhere the pellet should form. FIG. 65C shows the pellet is retainedthroughout the experiment. FIG. 66 shows the impact of the agitationrate on pellet persistence by overlaying the SERS curves from the 2 Hzand 1 Hz agitation rates (FIGS. 65A and 65C, respectively). With sloweragitation, the signal decay is much slower and the peak is much broaderthan when agitation is fast. Furthermore, real-time monitoring of thepellet through an in-line camera indicates that the loss of signal forfast agitation is due to the absence of the pellet, while for the slowagitation the pellet is always formed. The pellet fails to form near 3hours at 2 Hz agitation, but is always present using 1 Hz agitation.Consistent formation of the pellet at high organism load leads to longerpersistence of the SERS signal. This persistence in the SERS signal maybe advantageous if, for example, there is a delay between when thesample is added to the detection reagents and when the sample is placedinto the instrument.

Example 11 Determination of the Presence of a Targeted Pathogen UsingVisual Inspection of the Pellet in Sandwich Immuno-Assays

In this example a method for detection of microorganisms within amicrobiological sample that can eliminate the need for laser, optics,and spectrometer according to an embodiment of the invention isdescribed. This method involves the use of a camera to capture imagesduring reads in order to monitor the formation of a pellet during thecourse of a SERS-HNW assay.

In the experiment described in example 11 and shown in FIG. 65A, 65B,and 65C, the presence of the microorganism causes the pellet to grow insize (FIGS. 65A and 65C). In contrast, when the microorganism is absent,both the pellet size and the SERS signal remain stable. Without thepresence of the targeted pathogen, no sandwiches can be formed,resulting in no Raman signal and no increase in pellet size. FIG. 67shows the pellet size for a negative sample compared to the pellet sizefor a positive sample after 3 hours of secondary enrichment in acarousel system (see e.g., FIG. 24). This figure shows a larger pelletformed for the positive sample compared to the negative sample.

During secondary enrichment of a sample which contains conjugated SERStags and magnetic beads and the targeted pathogen, images show thatpellet size increases, and in some cases, fails to form as the assayprogresses. The growth in pellet size and/or disappearance of the pelletis an indication of the presence of the targeted pathogen. Imagescaptured during reads of samples that contain conjugated SERS tags andmagnetic beads with no pathogen show no change in pellet size and nopellet disappearance. Using image analysis to monitor pellet size maypresent a method of detecting microorganisms in the assay. This methodof detection can be used alone or in conjunction with another detectionmethod.

Example 12 Real-Time Detection of E. coli O157:H7 During Culture in FoodSamples

FIGS. 68A, 68B and 68C show representative data acquired with a carouselsystem (see e.g., FIG. 24) for the detection of E. coli 0157 instomached ground beef, spinach rinsate, and milk solids.

Raw ground beef was prepared according to the USDA MicrobiologyLaboratory Guidebook (MLG Chapter 5). 25 g samples of ground beef werediluted with 225 ml mTSB with Novobiocin in a stomacher bags. Eachstomacher bag was then stomached in a Seward Stomacher® 400 for 2minutes. 5 ml aliquots of the stomached ground beef were transferred totubes containing SERS tag and magnetic particle conjugates. E. coliO157:H7 (ATCC 43888) was grown in an overnight culture in Nutrient Brothfrom a single colony at 37° C. in a shaking culture. The culture wasserially diluted down to approximately 10²-10⁴ in Nutrient Broth. A 0.05ml aliquot was added to each positive tube and a 0.05 ml aliquot ofNutrient Broth was added to negative control tubes.

The spinach rinsate sample was prepared according to the FDABacteriological Analytical Manual (BAM Chapter 4A). An equal weight ofButterfield's phosphate buffer was added to spinach leaves in are-sealable plastic bag and agitated by hand for 5 minutes. The spinachrinsate was then added to an equal volume of double strength (×2) mBPWp.E. coli O157:H7 (ATCC 43888) was grown in an overnight culture inNutrient Broth from a single colony at 37° C. in a shaking culture. Theculture was serially diluted and inoculated into the spinach rinsate+(×2) mBPWp at a concentration of 103 or 0 cfu/ml. 5 ml aliquots ofthese samples were added to tubes containing SERS tag and magneticparticle conjugates.

The milk sample was prepared according to the FDA BacteriologicalAnalytical Manual (BAM Chapter 4A). Whole milk was centrifuged for 10minutes at 10,000×g. The supernatant layer was poured off and the pelletwas resuspended in mBPWp at 1.125 times the original milk volume. E.coli O157:H7 (ATCC 43888) was grown in an overnight culture in NutrientBroth from a single colony at 37° C. in a shaking culture. The culturewas diluted down to 5000 cfu/ml in Nutrient Broth. 50 ul aliquots of thediluted E. coli O157:H7 culture or Nutrient Broth (negative control) wasadded to 5 ml tubes of the resuspended milk culture plus assay reagents.

All inocula were plated for enumeration on BD BBL™ CHROMagar™ plates.Tubes were inserted into the carousel system for real time monitoringduring growth at 35° C. for 8 hours.

Example 13 Real-Time Detection of Salmonella During Culture in FoodSamples

FIG. 69 shows an example in which heat stressed S. Enteritidis (ATCC13076) was detected during real-time growth in ground beef plus culturemedia. Salmonella Enteritidis was grown in an overnight culture inNutrient Broth from a single colony at 37° C. in a shaking culture. Theculture was diluted down 1:100 in Nutrient Broth and heat stressed for20 minutes at 54° C. The heat stressed sample was further diluted downto 200 cfu/ml in Nutrient Broth. Two 25 g samples of raw ground beefwere inoculated with 1 ml aliquots of the diluted, heat stressed cultureof S. Enteritidis. The inoculum was plated for enumeration on NutrientAgar plates. The inoculated ground beef samples were hand massaged in astomacher bag for 2 minutes to thoroughly mix the inoculum. 225 ml ofSDIX RapidChek® Salmonella SELECT™ Primary Media with supplement wereadded to the inoculated samples in the stomacher bag. Negative controlsamples were prepared with 25 g of raw ground beef in 225 ml of SDIXRapidChek® Salmonella SELECT™ Primary Media with supplement. Eachstomacher bag was then stomached in a Seward Stomacher® 400 for 2minutes. The stomacher bags were then placed in a 42° C. incubator forapproximately 22 hours. 100 ul samples of the enriched primary cultureswere then added to tubes containing 4.5 ml SDIX Salmonella SecondaryMedia, 0.4 ml SDIX RapidChek® Salmonella SELECT™ Primary Media and assayreagents. The tubes were then inserted into the carousel system (seeFIG. 24) for real time monitoring during growth at 42° C. for 8 hours.Positive samples were detected within approximately 2 hours, whilenegative samples resulted in flat detection curves.

Example 14 Detection of Listeria in an Environmental Sample

FIG. 70 shows the detection of Listeria monocytogenes (ATCC 19115) on astainless steel coupon. L. monocytogenes (ATCC 19115) was grown in anovernight culture in Brain Heart Infusion Broth from a single colony at30° C. in a shaking culture. The culture was diluted down to 5×10⁴ or 0cfu/ml in PBS+5% milk. A 0.1 ml aliquot was placed on a 1″×1″ stainlesssteel coupon and allowed to air dry overnight. The next day, cottontipped swabs, wet with D/E neutralization broth, were swiped across thesurface several times. The swabs were then added to 5 ml of SDIXListeria media with supplement, SERS tags and magnetic particles intubes. The tubes were then inserted into the carousel system (see FIG.24) for real time monitoring during growth at 30° C. Positive sampleswere detected between approximately 19 and 27 hours, while negativesamples resulted in flat detection curves.

Example 15 Detection of Salmonella Using Flat-Bed System

In this example Salmonella was detected using linear agitation and aflat-bed system (see e.g., FIG. 25). Salmonella Typhimurium (ATCC 14028)and Salmonella Enteritidis (ATCC 13076) were grown separately inovernight cultures in SDIX RapidChek® Salmonella SELECT™ Primary Mediawith supplement at 42° C. A 1:100 dilution of each strain was made intoseparate SDIX Salmonella Secondary Media lots. The starting inoculationfor each strain in secondary media was determined to be 1×10⁷ CFU/mL byplate count on Nutrient agar plates. Each strain was inoculated induplicates into tubes containing SERS tags and magnetic particlesconjugated with anti-Salmonella antibodies (Virostat 0701). These tubes,along with two negative samples (SDIX secondary media and conjugatedSERS tags and magnetic particles with no Salmonella) were placed in theinstrument for monitoring during secondary enrichment at 42° C.

The system used in this example was a flat-bed configuration (see e.g.,FIG. 25). In this configuration, agitation is by linear reciprocationalong the axis of the tubes, which may be programmed for differentfrequencies and profiles throughout the assay. Each cycle consists ofthe following phases: mixing, pre-pellet dispersion, pelleting, reading,and dispersing. The magnet configuration used in this example was asingle bar magnet with N-pole facing the samples. Once a pellet wasformed, the bar was moved away from the samples to allow reading. FIG.71 shows the agitation frequency and throw used for each phase. Theexperiment was run for ˜19 hours with a cycle repeating every ˜20minutes.

The pre-pellet dispersion phase is intended to re-suspend settled solidin the SDIX secondary media prior to pelleting. Settled solid from themedia is known to interfere with pelleting of magnetic complexes. Thesingle bar magnet is brought in contact with the tubes during agitationand the samples are pelleted for 60 seconds. Agitation is stopped for 5seconds and the magnet is moved away from the sample tubes to allow theoptics engine to interrogate each pellet. A camera also captures imagesof each pellet. The agitation resumes to disperse the pellet and thecycle repeats.

FIG. 72 shows the SERS curves during secondary enrichment of S.Typhimurium (ATCC 14028), S. Enteriditis (ATCC 13076), and negativesamples. As can be seen, SERS curves are smooth and can easily bedistinguished from the negatives. FIG. 73 shows images of the pelletsformed at various times during secondary enrichment of S. Typhimurium.As can be seen, round, dense pellets are consistently formed throughoutthe assay using the flatbed instrument.

Example 16 Linear Versus Rocking Agitation

In this example, identical Salmonella assays were run on two carouselsystems (see e.g., FIG. 24) using different agitation methods: a linearreciprocation along the axis of the tubes and a rocking oscillation.Salmonella Enteritidis (ATCC 13076) and Salmonella Kentucky (ATCC 9263)were grown separately in overnight cultures in SDIX RapidChek®Salmonella SELECT™ Primary Media with supplement at 42° C. A 1:100dilution of each strain was made into separate SDIX Salmonella SecondaryMedia lots. The starting inoculation for each strain in secondary mediawas determined to be 1×10⁷ CFU/mL by plate count on Nutrient agarplates. Each strain was inoculated in duplicate into tubes containingSERS tags and magnetic particles conjugated with anti-Salmonellaantibodies from Virostat (0701). These tubes were placed in the carouselsystems for monitoring during secondary enrichment at 42° C. Eachinstrument read 2 times per hour, pelleted for 30 seconds, and agitatedat 1 Hz.

FIG. 74 shows SERS curves obtained from the rocking agitation and linearagitation carousel system during secondary enrichment of S. Enteriditisand S. Kentucky. It can be seen that good results are obtained with bothmethods.

In this example, linear agitation resulted in some advantages over therocking motion. Pelleting performance was better using linear agitationcompared to rocking because the pellet was always formed at the centerof the read head using linear agitation. The rocking agitation systemdoes not oscillate symmetrically about the rocker arm, causing the wheelof tubes to favor the forward motion. This asymmetric fluid motioncauses the fluid force on the pellet to favor the front side of thetubes. Due to its mechanical simplicity compared to rocking agitation,linear agitation is a preferred method of agitation.

Example 17 Compatibility of Real Time No Wash Assay with SubsequentSample Processing

In this example, the compatibility of the SERS-based real time assaywith sample processing tests that are typically performed followingdetection of a positive blood culture sample by conventional gas sensorswas tested. These tests may be used to provide organism identificationout of a positive blood culture bottle. These tests include standardtube coagulase assays, latex agglutination assays, gram staining,chromogenic media development, manual antibiotic susceptibility testingand anti-fungal inhibition on plated cultures.

The standard tube coagulase assays were performed by separatelyselecting several colonies of S. aureus or S. epidermidis from a streakplate and emulsifying them into BACTEC™ media. A 50 μl sample ofemulsified bacteria with or without SERS reagents (at assayconcentrations) was added to 500 μl of EDTA rabbit plasma and incubatedat 37° C. The S. aureus samples with and without SERS reagents bothcoagulated the plasma within 4 hours (FIG. 75). The S. epidermidissamples did not coagulate the rabbit plasma within 4 hours. Therefore,the presence of SERS reagents does not impede the ability to distinguishS. aureus from S. epidermidis via coagulase activity, even at relativelyhigh SERS reagent concentrations.

A latex agglutination test for S. aureus identification was alsoevaluated for any interference caused by the SERS assay particles. S.aureus and S. epidermidis samples with and without SERS reagents (atassay concentrations) were prepared as described above. One drop of BDBBL™ Staphyloslide™ test latex was then added to the assay card, as wasone drop of control latex. To each type of latex, 10 μl samples of 1) S.epidermidis with SERS reagents, 2) S. epidermidis without SERS reagents,3) S. aureus with SERS reagents, and 4) S. aureus without SERS reagentswere added. The solutions were mixed and rocked for ˜20 sec. FIG. 76shows the cards with S. epidermidis (left card) and S. aureus type 8(right card). Bacterial samples with SERS reagents were added to the toprow, while samples without SERS reagents were added to the bottom row.The results are identical for samples with and without reagents, withonly the S. aureus samples showing agglutination. The only samples toshow agglutination were the S. aureus samples with and without SERSreagents, demonstrating the SERS reagents do not impede latexagglutination in the presence of S. aureus, do not falsely agglutinatecontrol latex, and do not falsely agglutinate test latex in the presenceof S. epidermidis.

Gram staining with assay reagents was also performed as a test ofdownstream processing compatibility. SERS tags and magnetic particles inbuffer were added to BD BBL Control Gram Slides containingStaphylococcus aureus ATCC 25923 (gram positive cocci) and Escherichiacoli ATCC 25922 (gram negative rods) and imaged using a 100× oilimmersion objective, as is typically used in the clinic. FIG. 77 showsthe magnetic particles and SERS tags without the control organisms. Themagnetic particles are clearly visible as large brown spheres. Themagnetic particles are also uniform in color and size, effectivelyserving as an internal size standard (˜3 μm) for microscopy. The SERStags, which are 0.1-0.2 μm in diameter, are not visible. FIG. 78 shows amagnetic particle in the presence of a mixture of S. aureus (purplecocci) and E. coli (pink rods) imaged at 100×. Magnetic particles areclearly unstained and easily distinguishable from the microorganisms inthis image.

CHROMagar™ chromogenic media allows identification, differentiation andseparation of single pathogen by a single color developed in the solidmedia. Samples from overnight blood cultures of S. aureus type 8 and S.epidermidis containing SERS reagents were streaked onto CHROMagar™plates. The results we obtained (FIG. 79) indicate that the SERSreagents do not impact the ability to obtain single colonies and do notimpede the species-specific CHROMagar™ color development, wherein S.aureus is shown on the left and S. epidermidis is shown on the right. Asexpected, S. aureus colonies are mauve while S. epidermidis colonies arewhite when streaked on BD BBL™ CHROMagar™ Staph aureus plates.

Manual antibiotic testing using the agar disc diffusion method (BDSensi-disc™) was also tested in the presence of SERS reagents. Overnightblood cultures of E. coli O157 with SERS reagents were streaked on BDBBL™ Mueller Hinton II Agar plates and three BD BBL™ Sensi-disc™ testdiscs were placed on top and the culture allowed to grow at 37° C.overnight. The next day, the zones of inhibition (FIG. 80) were measured(in mm) and compared to the Sensi-disc™ Zone Diameter Interpretive Chartfor the determination of sensitive, inhibitory or resistant isolates.FIG. 80 shows Ampicillin-10 (top left), Levofloxacin-5 (top right),Vancomycin-30 (bottom)). The zone diameter measurements did not varymore than 1-2 mm between the E. coli culture with reagents and theculture without reagents (FIG. 81). This process was repeated with otherblood culture bacteria and yeast (Table 82), which shows that theability to determine the antibiotic susceptibility of a microorganismusing the disc diffusion method is not impacted by the presence of SERSreagents.

For testing yeast, Nystatin Taxo™ discs were used. These discs are notused for susceptibility testing, but for differentiation and isolationof bacteria from specimens with both bacteria and yeast. Therefore, aslightly different method was tested. Mixed blood cultures of E. coliand C. albicans with and without SERS reagents were streaked onto TSA IIplates. The Nystatin Taxo™ discs were placed on top and the cultureswere grown at 37° C. overnight. In both samples with (left image of FIG.83) and without (right image of FIG. 83) assay reagents, the Nystatininhibition of C. albicans growth resulted in areas of isolated E. colicolonies.

Example 18 Effect of Agitation Frequency and Pelleting Time on PelletFormation

This example pertains to pelleting using a configuration where the tubeis coupled to the magnet (see e.g., FIG. 49) such that the magnet moveswith the tube and is held in the same relative position to the tubethroughout the agitation. In a series of experiments, SERS tags werecovalently linked to tosyl-activated magnetic particles to form aSERS-magnetic bead precomplex (PC). Pre-complexed beads are prepared bycovalent linkage of SERS particles to Dynabeads® M-280 Tosyl-activatedmagnetic particles through reaction of thiol(-SH) groups on the SERSsurface with tosyl (Tos) groups on the surface of the magnetic particlesPC acts as a model system for pellet formation testing where the pelletcan be interrogated for SERS signal. In this example, pellet formationof PC in a commercial secondary media for Salmonella (SDIX RapidChek®Salmonella SELECT™) was compared using a variety of agitationfrequencies and pelleting times. These tests were performed using aflat-bed system configuration (see e.g., FIG. 25) with a single barmagnet with the N-pole facing the tubes (see e.g., FIG. 52).

FIG. 84 shows a table with images of pellets formed using PC in SDIXSalmonella secondary media using three different agitation frequenciesand three different pelleting times. PC in SDIX secondary media waspelleted by agitating the tubes coupled with the bar magnet at varyingagitation frequencies at 50 mm throw (amplitude). Agitation was stoppedand the magnet was allowed to persist for 5 seconds before moving thebar magnet away from the tubes. Images of the pellets were captured oncethe bar magnet was moved away from the tubes.

As shown in FIG. 84, fast agitation forms a denser pellet compared toslow agitation. This is likely due to the solid media settling usingslow agitation and interfering with pellet formation. Using fastagitation, the solid is suspended in solution and magnetic complexes canbe pulled into a pellet with less interference from the media. It can beseen in the pellets formed using 0.7 Hz agitation frequency with 30, 60,and 90 second pelleting times that settled media interferes with theability to pull a dense pellet, as evidenced by the diffuse pellet.

Example 19 Effect of Agitation Frequency on Pellet Dispersion

This example pertains to measuring the time required to fully disperse apellet using a variety of agitation throws (amplitude) and frequencies.In each case, a pellet was formed using a configuration in which thetube is coupled to the magnet (see e.g., FIG. 49) such that the magnetmoves with the tube and is held in the same relative position to thetube throughout the agitation. The magnet used in this example was asingle bar magnet with the N-pole facing the sample tubes, such as shownin FIG. 52.

In this example, the tube was manually shaken before each test tothoroughly mix the media (SDIX RapidChek® Salmonella SELECT™) and PC.The sample was loaded into the flatbed instrument and a pellet wasformed by agitating at 1.8 Hz and 25 mm throw for 90 seconds. Agitationwas stopped and the magnet was allowed to persist for 5 seconds beforemoving the magnet bar away from the tubes. Various agitation frequenciesand throws were used in separate tests to disperse the pellet. Pelletdispersal was monitored by visual inspection and the time required tofully disperse the pellet was measured. Data with an asterisk indicatesthat no settled media was observed.

As shown in FIG. 85, fast agitation disperses the pellet in less timecompared to slow agitation. Also, for a given agitation frequency, thepellet disperses quicker with a longer agitation throw. Based onobservations in this example, solid media in the secondary media remainssuspended in solution at agitation frequencies above 2.5 Hz at 25 mmthrow and above 1.5 Hz at 50 mm throw.

Example 20 Fluorescence HNW Assay Feasibility Testing in the Presence ofFood

This example demonstrates the feasibility of conducting a homogeneous nowash assay in conjunction with culture using near infrared (“NIR”)fluorescent particles instead of SERS tags. In this example, fluorescentsilica nanoparticles were fabricated using a modified Stober growthtechnique incorporating both a silane-NIR dye conjugate (to provide thefluorescent signal) and a thiolated silane (to provide a chemical handlefor antibody conjugation). Particles were characterized by transmissionelectron microscopy (“TEM”), UV/Vis extinction spectroscopy, andfluorescence spectroscopy, and found to be relatively monodisperse andbright. FIG. 96 illustrates a TEM image of NIR fluorescent silicananoparticles (scale bar is 200 nm), while FIG. 97 illustrates afluorescence spectrum of NIR fluorescent nanoparticles (OD 0.5) andRaman spectrum of standard ES/HB SERS tags (OD 1.2). Fluorescentnanoparticles were conjugated with Listeria Ab using a standardconjugation protocol which was modified to account for differences influorescent nanoparticle concentration, surface area, and mass relativeto SERS tags. Conjugated fluorescent silica nanoparticles were tested ina Listeria HNW assay on a carousel system 150 (See FIG. 24) using 10%w/v blended samples of spinach and cabbage. Control tests were performedusing SERS tags with both spinach and cabbage samples.

Fluorescent tags were able to successfully detect Listeria in both foodsamples. FIG. 98 depicts the spinach data collected using NIRfluorescent nanoparticle tags and SERS tags, while FIG. 99 depicts thecabbage data collected using NIR fluorescent nanoparticle tags and SERStags. Both signal and background were found to be higher for fluorescenttags than for SERS tags, however, detection was successful withrelatively high signal to background ratios of ˜4:1.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims and equivalents thereof.

All publications, patent applications, patents, and other references areherein incorporated by reference to the same extent as if eachindividual publication, patent application, patent, and other referencewas specifically and individually indicated to be incorporated byreference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art.

The foregoing description is intended to be exemplary of variousembodiments of the invention. It will be understood by those skilled inthe art that various changes and modifications to the disclosedembodiments can be made without departing from the purview and spirit ofthe invention as defined in the appended claims.

1. A method for detecting one or more microorganisms in a sample, themethod comprising: (a) providing a sample suspected of containing one ormore microorganisms; (b) disposing said sample in an assay vial, whereinsaid assay vial has disposed therein a culture medium capable ofsupporting microorganism growth to form a culture sample and a reagentcomprising one or more indicator particles having associated therewithat least one specific binding member having an affinity for said one ormore microorganisms; and one or more magnetic capture particles havingassociated therewith at least one specific binding member having anaffinity for said one or more microorganisms, wherein the binding memberassociated with the indicator particles can be the same or differentthan the binding member associated with the magnetic capture particles;(c) incubating the culture sample for a predetermined period of time toform a magnetic capture particle-microorganism-indicator particlecomplex if said one or more microorganisms are present in the sample;(d) agitating the assay vial; (e) exposing said magnetic captureparticle-microorganism-indicator particle complex to a magnetic field toinduce said complex to migrate to a localized area of said assay vial;(f) optically interrogating said localized area of said assay vial toinduce said indicator particle to produce a detectable signal to detectsaid one or more microorganisms in said sample; (g) dispersing saidmagnetic capture particle-microorganism-indicator particle complex; and(h) repeating steps (c)-(g) one or more times.
 2. The method accordingto claim 1, wherein repeating steps (c)-(g) occurs in regular timeintervals.
 3. The method according to claim 1, wherein steps (c) and (d)occur concurrently.
 4. The method according to claim 1, wherein step (e)forms a pellet comprising magnetic captureparticle-microorganism-indicator particle complexes.
 5. The methodaccording to claim 4, wherein the pellet is detectable using visual oroptical means.
 6. The method according to claim 1, wherein the samplecomprises a blood sample.
 7. The method according to claim 1, whereinthe sample comprises a food sample.
 8. The method according to claim 1,wherein the sample comprises an environmental sample.
 9. The methodaccording to claim 1, wherein the sample comprises an agriculturalsample.
 10. The method according to claim 1, wherein disposing saidsample in an assay vial comprises transferring a desired amount of saidsample, along with optional culture media, from a vessel, wherein thevessel comprises: a container for receiving a culture sample therein,the container having an open end and a closed end; a lid configured toengage the open end of the container in a fluid-tight connection; abasket coupled to the lid and including at least one reservoir, thebasket being disposed between the open end and the closed end of thecontainer, the reservoir configured to hold a volume of culture sampletherein; and at least one needle assembly engaged with the lid, theneedle assembly including a needle extending within the reservoir,wherein the needle is configured to selectively withdraw a samplecontained in the reservoir, and wherein the needle is further configuredto engage a vial for a biocontained transfer of the sample from thereservoir to the vial.
 11. The method according to claim 1, whereindisposing said sample in an assay vial comprises extracting the sample,along with optional culture media, in a biocontained fashion from avessel in which the magnetic particle and indicator particles are notpresent.
 12. A vessel for metering a desired amount of a sample, thevessel comprising: a container for receiving a sample therein, thecontainer having an open end and a closed end; a lid configured toengage the open end of the container in a fluid-tight connection; abasket coupled to the lid and including at least one reservoir, thebasket being disposed between the open end and the closed end of thecontainer, the reservoir configured to hold a volume of sample therein;and at least one needle assembly engaged with the lid, the needleassembly including a needle extending within the reservoir, wherein theneedle is configured to selectively withdraw a sample contained in thereservoir, and wherein the needle is further configured to engage a vialfor a biocontained transfer of the sample from the reservoir to thevial.
 13. The vessel according to claim 12, wherein the container isconstructed of a material transparent to visible radiation.
 14. Thevessel according to claim 12, wherein the basket defines a firstreservoir and a second reservoir, and wherein the lid defines a firstneedle assembly with a first needle extending within the first reservoirand a second needle assembly with a second needle extending within thesecond reservoir.
 15. The vessel according to claim 14, wherein thefirst needle and the first reservoir are configured for use with a firsttype of assay, wherein the second needle and the second reservoir areconfigured for use with a second type of assay, and first type of assayis different than the second type of assay.
 16. The vessel according toclaim 15, wherein the first reservoir is configured to holdapproximately 5 mL, and wherein the second reservoir is configured tohold approximately 100 μL.
 17. The vessel according to claim 12, whereinthe basket defines a rib that is configured to aid in draining of fluidthrough the basket.
 18. The vessel according to claim 12 furthercomprising a head space above the at least one reservoir to enable thesample to readily enter the at least one reservoir when the container istilted.
 19. The vessel according to claim 18, wherein the basket definesat least one hole, wherein the hole is configured to enable excesssample to drain into the container after the container is turned back toan upright position.
 20. The vessel according to claim 12, wherein thelid defines a vent for allowing any nonhazardous, gaseous byproducts toescape form the container to prevent pressure build up within thecontainer.
 21. The vessel according to claim 12 further comprising avent post extending from a bottom surface of the lid, wherein the ventpost defines an opening therethrough for receiving and engaging a filterfor filtering gaseous byproducts exiting the container.
 22. The vesselaccording to claim 12 further comprising a first engagement postextending outwardly from a bottom surface of the lid and a secondengagement post extending outwardly from an upper surface of the basket,wherein the first engagement post is configured to align with and engagethe second engagement post.
 23. The vessel according to claim 12,wherein the lid defines at least one keyed opening that is configured toallow mating of a specific cap of the vial.
 24. The vessel according toclaim 12, wherein the needle comprises a protective sleeve that coversthe needle, wherein the protective sleeve is configured to be compressedas the vial is pushed downwardly and into engagement with the needle,and wherein the protective sleeve is configured to return to itsoriginal shape to cover the needle as the vial is withdrawn fromengagement with the needle in order to enable the biocontained transfer.25. The vessel according to claim 12, wherein the lid further comprisesat least one back-off feature to prevent unscrewing of the lid withoutadditional disengagement of the back-off feature.
 26. A lid assembly foruse with a container for metering a desired amount of a sample, the lidassembly comprising: a lid configured to engage the open end of acontainer in a fluid-tight connection, the lid optionally containing agasket; a basket coupled to the lid and including at least onereservoir, the reservoir configured to hold a volume of sample therein;and at least one needle assembly engaged with the lid, the needleassembly including a needle extending within the reservoir, wherein theneedle is configured to engage a vial for a biocontained transfer of asample from the reservoir to the vial.
 27. The lid assembly according toclaim 26, wherein the basket defines a first reservoir and a secondreservoir, and wherein the lid defines a first needle assembly with afirst needle extending within the first reservoir and a second needleassembly with a second needle extending within the second reservoir. 28.The lid assembly according to claim 27, wherein the first needle and thefirst reservoir are configured for use with a first type of assay,wherein the second needle and the second reservoir are configured foruse with a second type of assay, and first type of assay is differentthan the second type of assay.
 29. The lid assembly according to claim28, wherein the first reservoir is configured to hold approximately 5mL, and wherein the second reservoir is configured to hold approximately100 μL.
 30. The lid assembly according to claim 26, wherein the basketdefines a rib that is configured to aid in draining of fluid through thebasket.
 31. The lid assembly according to claim 26, wherein the liddefines a vent for allowing any nonhazardous, gaseous byproducts toescape.
 32. The lid assembly according to claim 26 further comprising avent post extending from a bottom surface of the lid, wherein the ventpost defines an opening therethrough for receiving and engaging a filterfor filtering gaseous byproducts.
 33. The lid assembly according toclaim 26 further comprising a first engagement post extending outwardlyfrom a bottom surface of the lid and a second engagement post extendingoutwardly from an upper surface of the basket, wherein the firstengagement post is configured to align with and engage the secondengagement post.
 34. The lid assembly according to claim 26, wherein thelid defines at least one keyed opening that is configured to allowmating of a specific cap of a vial.
 35. The lid assembly according toclaim 26, wherein the needle comprises a protective sleeve that coversthe needle, wherein the protective sleeve is configured to be compressedas a vial is pushed downwardly and into engagement with the needle, andwherein the protective sleeve is configured to return to its originalshape to cover the needle as a vial is withdrawn from engagement withthe needle in order to enable the biocontained transfer.
 36. The lidassembly according to claim 26, wherein the lid further comprises atleast one back-off feature to prevent unscrewing of the lid withoutadditional disengagement of the back-off feature.
 37. A detection vialfor a biocontained transfer of a sample from a reservoir to the vial,wherein the detection vial comprises: a body with an opening; and a capfor engaging the body and sealing the opening, wherein the cap comprisesa stopper configured to retain a vacuum within and, optionally, anabsorbent pad, wherein the cap defines a plurality of ribs configured tofit within a keyed opening of a lid of a vessel.
 38. The detection vialaccording to claim 37, wherein the body defines a constant wallthickness along a length so as to enhance pelleting and opticalanalysis.
 39. A detection vial for biocontained transfer of a samplefrom a reservoir, wherein the detection vial comprises: a body with anopening; and a cap for engaging the body and sealing the opening,wherein the cap comprises a stopper configured to retain a vacuum withinand, optionally, an absorbent pad, wherein the cap may be configured tofit a specified transfer port.
 40. The detection vial according to claim39, wherein the body defines a constant wall thickness along a length soas to enhance pelleting and optical analysis.
 41. A syringe for abiocontained transfer of a sample from a reservoir to the syringe,wherein the syringe comprises: a body; a handle; a plunger rod coupledto the handle; a cap; a plunger coupled to an end of the plunger rod andconfigured to be longitudinally displaced within the body; a septum; anda seal.
 42. A system for automatically processing a plurality of tubescontaining a culture sample, said system comprising: an incubator forreceiving a plurality of sample tubes therein, the incubator configuredto incubate the sample tubes at a predetermined temperature; a firsttranslational device coupled to the sample tubes and configured to movethe sample tubes for agitating the sample tubes, the first translationaldevice further configured to move the sample tubes from the incubator toa detection zone and to agitate the sample tubes within the detectionzone; a magnet assembly configured to apply a magnetic field to theplurality of sample tubes within the detection zone; an optical deviceconfigured to interrogate each of the plurality of sample tubes withinthe detection zone for detecting one or more microorganisms; and asecond translational device coupled to the optical device and configuredto move the optical device within the detection zone for interrogatingeach of the sample tubes.
 43. The system according to claim 42, whereinthe magnet assembly is configured to pivot away from the sample tubesfor interrogation by the optical device.
 44. The system according toclaim 42, wherein the system defines a plurality of thermal zones thatcan operate at different temperatures, wherein each thermal zone isconfigured to contain one or more incubators.
 45. The system accordingto claim 42 further comprising at least one heating element configuredto heat the incubator.
 46. The system according to claim 42, wherein theincubator is configured to receive a tray for holding a plurality ofsample tubes.
 47. The system according to claim 46, wherein the firsttranslation device is configured to oscillate the tray horizontallyalong an axis of the incubator.
 48. The system according to claim 42,wherein the magnet assembly comprises a pair of longitudinal magnetsspaced apart, and wherein the optical device comprises a read headextending between the pair of longitudinal magnets.